腸管上皮LAT1によるパネート細胞数修飾とApc[Min/+]マウスの腫瘍形成への影響の検討

概要

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PDF issue: 2024-05-02

LAT1 expression influences Paneth cell number
and tumor development in Apc[Min/+] mice

隋, 云⻰
(Degree)
博士（医学）

(Date of Degree)
2023-03-25

(Resource Type)
doctoral thesis

(Report Number)
甲第8682号

(URL)
https://hdl.handle.net/20.500.14094/0100485866
※ 当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。

（課程博士関係）
学位論文の内容要旨

LAT1 expression influences Paneth cell number and tumor development
in ApcMin/+ mice

腸管上皮 LAT1 によるパネート細胞数修飾と ApcMin/+
マウスの腫瘍形成への影響の検討

神戸大学大学院医学研究科医学科専攻
消化器内科学
指導教員：児玉裕三教授
隋云龙

Introduction
Epidemiological studies show that the incidence of intestinal tumors in both small and
large intestines is increasing. It also suggests that increased intake of red or processed
meat is associated with those increased incidences. Amino acids, which are metabolized
from those foods, are not only required to build body structures but also act as signaling
molecules in cells to modulate signaling pathways. Although essential to physiological
function, excessive levels of amino acids can contribute to prevalent diseases, such as
heart failure and cancer. However, the mechanism by which those nutrients contribute to
the incidence of intestinal tumors is not fully elucidated.
L-type amino acid transporter 1 (LAT1) transports a wide range of essential amino
acids. It is known to be only expressed in some normal tissues, such as endothelial cells
and placenta, but absent in many normal tissues, such as the pancreas, breast, and lung;
however, it becomes highly expressed in tumors originating from these organs. LAT1 is
also detected in human intestinal tumors and associated with accelerated tumor cell
proliferation. However, whether LAT1 plays any other role in intestinal tumorigenesis is
not well understood.
Adenomatous polyposis coli (APC) is a tumor-suppressor gene, and its mutation causes
aberrant activation of the Wnt/β-catenin pathway that promotes tumor growth. A mouse
strain ApcMin/+ is a widely used murine model of intestinal tumors which develops from
the intestinal stem cells. In our study, we generated intestinal epithelium-specific LAT1
deficient ApcMin/+ mice to investigate the role of LAT1 in intestinal tumorigenesis.

Methods
Mice carrying LAT1 floxed alleles that also expressed Cre recombinase from the
promoter of gene encoding Villin were crossed to establish a strain which is LAT1
deficient only in the intestinal epithelium (LAT1fl/fl; vil-cre). It was further crossed to an
ApcMin/+ background (LAT1fl/fl; vil-cre; ApcMin/+), and those mice were analyzed for the
tumor phenotype.
Results
1. LAT1 was constitutively expressed in intestinal crypts: conditional deletion of
LAT1 led to fewer Paneth cells in the normal small intestine.
It has been widely recognized that LAT1 is expressed in the intestinal tumor cells,
however, it was not well studied whether it is expressed in the normal epithelium and
plays any physiological role. To determine whether LAT1 deletion affected steady state
intestinal structure, detailed histological analysis was performed using LAT1fl/fl; vil-cre
and LAT1fl/fl mice. Using hematoxylin and eosin staining, we confirmed that the colon
tissue structure was comparable between LAT1fl/fl; vil-cre and LAT1fl/fl mice; however,
the number of Paneth cells, which is only found in the small intestinal crypts, was
significantly reduced in LAT1fl/fl; vil-cre mice. This phenotype should not be observed if
LAT1 is only expressed in the intestinal tumor cells; thus, we conducted
immunohistochemistry for LAT1 on normal intestinal tissue sections. It was confirmed
that crypt base cells in both the small intestine and colon were LAT1 positive. These
results suggest that LAT1 is constitutively expressed in the crypts at the steady state and
affects the development of Paneth cells in the small intestine.

2. Conditional deletion of LAT1 in the intestinal epithelium reduced tumor number
and size in the small intestine but not in the colon of ApcMin/+ mice
To investigate whether LAT1 deficiency in the epithelium can affect tumorigenesis in
the ApcMin/+ mice, tumor phenotype between LAT1fl/fl; vil-cre; ApcMin/+ mice and
LAT1fl/fl; ApcMin/+ were compared. The result showed that LAT1fl/ fl; Villin-Cre; ApcMin/+
developed significantly fewer and smaller tumors in the small intestine compared to the
LAT1 sufficient control LAT1fl/fl; ApcMin/+ mice, but the colon showed no difference
between these two groups.
3. Conditional deletion of LAT1 suppressed the mTORC1 pathway and caused
apoptosis in tumors
To investigate the mechanism by which LAT1 deletion results in smaller and fewer
tumors in the small intestine, we performed Ki67 and TUNEL staining to analyze the
status of cell proliferation and apoptosis in the tumors, respectively. LAT1fl/fl; vil-cre;
ApcMin/+ tumor tissues exhibited a lower number of Ki67-positive cells and a higher
number of apoptotic cells, suggesting that deletion of LAT1 suppresses cell proliferation
and promotes apoptosis. mTORC1 pathway plays an important role in cell proliferation
and growth and is also shown to be activated downstream of LAT1. To investigate the
involvement of mTORC1 pathway, the phosphorylation of 4E-BP1 and S6K1 was
examined by western blotting using tumor tissues. The result showed that levels of
phosphorylation of both proteins were reduced. Furthermore, it showed the increase of
caspase-3 cleavage in LAT1fl/fl; vil-cre; ApcMin/+ tumors which supports the increased

number of apoptotic cells. Those data suggest that conditional deletion of LAT1 blocks
the tumor cell proliferation through reduced activation of mTORC1 pathway, and
promotes tumor cell apoptosis.
4. Organoids derived from LAT1-deleted crypts displayed fewer and smaller
spherical (tumor) organoids by reducing Wnt and mTORC1 pathways.
To confirm those in vivo findings using in vitro system, organoids derived from the
intestines of those mice were analyzed. The result showed that the crypts from LAT1fl/fl;
vil-cre; ApcMin/+ mice formed significantly fewer and smaller spherical organoids
compared to LAT1fl/fl; ApcMin/+ crypts, which recapitulated the in vivo phenotypes. We
next examined the phosphorylation of 4E-BP1 and S6K1 using protein extracted from the
organoids, and LAT1-deficient organoids showed a reduction of phosphorylation levels.
On the other hand, there was no clear promotion of caspase-3 cleavage. These data
suggest that LAT1 deletion directly affects the mTORC1 activation and slows down
spherical organoid growth, however, the promotion of apoptosis seems to be an indirect
effect only observed in vivo. To investigate the mechanism of fewer tumor numbers and
spherical organoids, we firstly speculated that LAT1 deficiency could reduce the number
of stem cell from which ApcMin/+ tumor originates. However, immunohistochemistry of
stem cells showed that the LAT1 deficiency did not affect its number in vivo. Next, as
Wnt pathway is critically involved in the tumorigenesis of ApcMin/+ mice, we speculated
that the modification of Wnt pathway activity is involved. Paneth cell is known to produce
Wnt3, therefore, Wnt3 expression levels were examined in the small intestinal tissue. As

expected, we confirmed that LAT1-deficient tissue showed a significantly reduced
amount of Wnt3. We also compared two other Wnts, Wnt6, and Wnt9b, which are also
shown to be expressed in the intestinal epithelium. Wnt3 showed the strongest expression
among those Wnts, and Wnt6 and Wnt9b levels were not affected by LAT1 deletion.
These data suggest that Wnt3 is the major Wnt affecting tumor initiation in our setting.
To further confirm the involvement of Wnt pathway, the expression of Wnt target genes
were investigated. Indeed, the expression levels of the target genes Ccnd1 and Axin2 were
significantly reduced, and c-Myc also showed a trend of reduced expression in LAT1fl/fl;
vil-cre; ApcMin/+-derived organoids compared with those in the LAT1fl/fl; ApcMin/+-derived
organoids. This indicated that Wnt pathway activation was indeed inhibited by LAT1
deficiency. Lastly, we confirmed exogenous Wnt3 supplementation to the culture
medium could recover the number but not the size of spherical organoids derived from
LAT1fl/fl; vil-cre; ApcMin/+ mice.
These data indicate that LAT1 expression in the small intestinal crypts plays a critical
role in tumor development through Wnt3 production from Paneth cells affecting the
activation of the Wnt/β-catenin pathway on a cell-extrinsic manner. However, once a
tumor develops, the mTORC1 pathway downstream of LAT1 supports the acceleration
of tumor growth in a cell-intrinsic manner.

Conclusion
Our findings showed that LAT1 is constitutively expressed in normal intestinal crypt
cells. Conditional deletion of LAT1 in the epithelium resulted in a reduced number of

Paneth cells and decreased Wnt3 production, while suppressing tumor development and
growth, which benefited ApcMin/+ mice with a small number of small-sized tumors. Our
findings indicate that the nutrient environment and the fate of intestinal tumors may be
connected, via amino acid transporter LAT1 expression.

神戸大学大学院医学（
系）
研究科（博士課程）
論 文審 査 の 結
甲第

3284号

氏

名

受 付 番号

果 の 要 旨

隋 云尤

腸管上皮 LATlによるパネー ト細胞数修飾 と Ap
c
Min／
＋
マウスの腫瘍
論文題目

1

形成への影響の検討

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1
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2
. Condi
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e
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t
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onofLATli
nt
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nt
e
st
inalep
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nves
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g
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ewhet
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rLATlde
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ici
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umor
i
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1t
heAp
cMi
n
/
+mi
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t
umorp
heno
t
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pebet
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l
/
f
l
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l
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r
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cM
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/
+mi
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cM
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+werecomp
a
r
e
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lt
s
howedt
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tLATl1
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1f
l
;Vi
li
n
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r
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v
+d
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o
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ant
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rt
umorsi
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inecomp
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r
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ot
heLA
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l LA
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m
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e be
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et
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.

3
. Condi
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e
l
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i
onof
LATlsupp
r
e
s
s
e
dt
hemTORClp
at
hwa
yandcausedapop
t
os
i
si
nt
umors
nv
e
s
t
i
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at
et
hemechani
smbywhi
chLATId
e
l
e
t
i
onr
e
s
u
lt
si
nsmalerandf
ewert
t
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m
o
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Toi
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o1
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t
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at
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i
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cmanner
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,onceat
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t
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up
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t
i
onof
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umorg
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hi
naceli
nt
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i
n
si
cmanner
.
ofLAT!s
Conclusi
on

o
1
di
ng
sshowedt
ha
tLAT!i
scons
t
i
t
ut
i
ve
l
yexp
r
e
s
s
e
di
nno
rmali
nt
e
s
t
i
nalcr
y
p
tcels
.Condi
t
i
onal
Ourf
t
ion ofLAT! i
nt
heep
i
t
hel
i
um r
e
s
u
lt
ed i
nareducednumberofPane
t
hcelsanddecreasedW n
t
3
del
e
p
roduc
t
ion,whi
l
es
up
p
r
e
s
si
ngt
umordevel
op
ment
andg
row
t
h,whi
c
hbene
f
i
t
e
dAp
cM
i
n
/
+mi
cewi
t
has
m
a
l
l
numbe
rof
s
mal
l
-s
i
z
edt
u
mors.Ourf
indi
ng
sJ
i
1
di
cat
et
ha
tt
henut
r
i
ent
e
nvi
r
onment
a
ndt
hef
a
t
eofi
nt
e
st
i
n
a
l
t
u
morsma
ybeconnect
ed,vi
aami
noaci
dt
r
a
ns
p
o
r
t
erLAT!exp
r
e
ss
i
o
n
.
本 研 究 は 、腸上皮 細 胞の腫瘍性変化 における LATlの役割を解 明したもので 、価値ある
業績である 。 よって 、本研究者は博士（ 医学） の学位 を得る資格があると認める 。