RESEARCH ARTICLE
Promotion of Colorectal Cancer Invasion and Metastasis through Activation of NOTCH–DAB1–ABL–RHOGEF Protein TRIO Masahiro Sonoshita 1 , Yoshiro Itatani 1,2 , Fumihiko Kakizaki 1 , Kenji Sakimura 3 , Toshio Terashima 4 , Yu Katsuyama 5 , Yoshiharu Sakai 2 , and M. Mark Taketo 1,2
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ABSTRACT We have recently identifi ed a metastasis suppressor gene for colorectal cancer:
AES / Aes , which encodes an endogenous inhibitor of NOTCH signaling. When Aes is
knocked out in the adenomatous epithelium of intestinal polyposis mice, their tumors become malig-
nant, showing marked submucosal invasion and intravasation. Here, we show that one of the genes
induced by NOTCH signaling in colorectal cancer is DAB1 / Dab1 . Genetic depletion of DAB1 suppresses
cancer invasion and metastasis in the NOTCH signaling–activated mice. DAB1 is phosphorylated by ABL
tyrosine kinase, which activates ABL reciprocally. Consistently, inhibition of ABL suppresses cancer
invasion in mice. Furthermore, we show that one of the targets of ABL is the RAC/RHOGEF protein
TRIO, and that phosphorylation at its Tyr residue 2681 (pY2681) causes RHO activation in colorectal
cancer cells. Its unphosphorylatable mutation TRIO Y2681F reduces RHOGEF activity and inhibits inva-
sion of colorectal cancer cells. Importantly, TRIO pY2681 correlates with signifi cantly poorer progno-
sis of patients with colorectal cancer after surgery.
SIGNIFICANCE: These results indicate that TRIO pY2681 is one of the downstream effectors of NOTCH
signaling activation in colorectal cancer, and can be a prognostic marker, helping to determine the
therapeutic modality of patients with colorectal cancer. Cancer Discov; 5(2); 198–211. ©2014 AACR.
See related commentary by Kranenburg, p. 115.
1 Department of Pharmacology, Graduate School of Medicine, Kyoto Univer-sity, Kyoto, Japan. 2 Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 3 Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan. 4 Department of Physiol-ogy and Cell Biology, Division of Anatomy and Neurobiology, Kobe University Graduate School of Medicine, Kobe, Japan. 5 Division of Developmental Neuro-science, Tohoku University Graduate School of Medicine, Sendai, Japan.
Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).
Current address for M. Sonoshita: Department of Developmental and Regenerative Biology, Icahn School of Medicine, Mount Sinai, New York, New York; current address for Y. Itatani: University of California, San Diego, Moores Cancer Center, La Jolla, CA.
Corresponding Author: M. Mark Taketo, Graduate School of Medicine, Kyoto University, Yoshida-Konoé-cho, Sakyo, Kyoto 606-8501, Japan. Phone: 81-75-753-4391; Fax: 81-75-753-4402; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-14-0595
©2014 American Association for Cancer Research.
INTRODUCTION The direct cause of cancer death is often its metastasis to
the vital organs. Metastasis is achieved through a multistep
cascade of events, and therefore ineffi cient as a whole ( 1 ).
Despite the efforts to fi nd mutations that are responsible for
metastasis, relatively few such genetic changes have been found
( 2 ), leading to the speculation that metastasis is driven by the
mechanisms for physiologic and/or pathologic body reactions.
The AES / Aes (Amino-terminal enhancer of split) gene
encodes the colorectal cancer metastasis suppressor AES that
functions as an endogenous inhibitor of NOTCH signal-
ing ( 3 ), which plays a variety of roles in cancer in a context-
dependent manner. In colorectal cancer cells, AES protein can
block the NOTCH signaling transcription complex composed
of the NOTCH intracellular domain (NICD), the transcrip-
tion factor RBPJ, and the cofactor MAML. Upon introduction
of homozygous Aes knockout mutation into the adenomatous
epithelium of Apc +/Δ716 intestinal polyposis mice, their tumors
become malignant due to NOTCH signaling activation, show-
ing submucosal invasion and intravasation ( 3 ). Consistently,
transendothelial migration (TEM) is increased signifi cantly,
when colorectal cancer cells with activated NOTCH signaling
are placed on an endothelial cell (EC) layer in culture. Thus,
reduced level of AES and stimulation of NOTCH signaling
are implicated in the invasion and intravasation of colorectal
cancer cells during metastasis ( 3, 4 ).
Although NOTCH signaling is involved in cell migration
during nervous system development ( 5 ), the precise molecu-
lar mechanisms remain to be elucidated. In the present study,
we have investigated how NOTCH signaling stimulates color-
ectal cancer metastasis. We have found coordinated activation
of NOTCH signaling in colorectal cancer via RBPJ-dependent
transcription of Dab1 , followed by ABL tyrosine kinase activa-
tion and phosphorylation of the RHOGEF protein TRIO.
RESULTS One of the Genes Induced by NOTCH Signaling–Dependent Transcription in Colorectal Cancer Cells Is DAB1/Dab1 , Which Promotes Metastasis
As we have shown recently, markedly invasive and intrava-
sating tumors develop in the intestines of compound mutant
mice for the Apc and Aes genes [ Apc +/Δ716 ;Aes Flox/Flox ;TgvCre ERT2
mice dosed with 4-hydroxytamoxifen (4-HT), abbreviated as
Apc −/− Aes −/− or Apc/Aes mice; ref. 3 ; Fig. 1A , left]. Although AES
inhibits NOTCH-dependent transcription in colorectal cancer
cells, it remains unclear how NOTCH signaling, activated by
loss of AES, stimulates progression of mouse tumors. We began
the present study with investigation on the role of the NOTCH
signaling transcription factor RBPJ. Notably, homozygous null
mutation of Rbpj reduced tumor invasion and intravasation
dramatically in Apc +/Δ716 ; Aes Flox/Flox ; Rbpj Flox/Flox ; TgvCre ERT2 mice
dosed with 4-HT ( Apc −/− Aes −/− Rbpj −/− or Apc/Aes/Rbpj ) without
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Sonoshita et al.RESEARCH ARTICLE
Figure 1. Transcription factor RBPJ is essential for invasion of mouse intestinal tumors and induces expression of DAB1/Dab1 as one of the down-stream genes. A and B, Rbpj gene knockout blocks invasion of intestinal tumors in Apc/Aes compound mutant mice. Note that after induction of intestinal epithelium– specifi c Cre recombinase–mediated mutagenesis, the tumor genotypes were Apc −/− Aes −/− and Apc −/− Aes −/− Rbpj −/− , respectively. The depth of tumor invasion was analyzed histopathologically by hematoxylin and eosin staining (A), and scored (B). Dotted lines in A, muscularis mucosae . Asterisk in A, a colorectal cancer gland invading into the submucosa. mu, intramucosal; sm, mp, and se, invasions reaching the submucosa, muscularis propria , and serosa, respectively ( n = 5 mice for each genotype). Apc/Aes/Rbpj mice showed extended life spans (6–7 months from ∼5 months). C, RBPJ target genes common to mouse and Drosophila . Mouse and ( fl y ) gene symbols. Data were extracted from ( 6, 7 ). D, binding sequence motifs for the NOTCH signaling transcription factor Su(H)/RBPJ/CBF1 in the fl y, mouse, and human promoter regions for the dab/Dab1/DAB1 genes, respectively. A pair of horizontal triangles; primers for the chromatin immunoprecipitation (ChIP) analysis in F. TSS, transcription start site. E, recombinant DLL4 (rDLL4) ligand induces DAB1 in cultured colorectal cancer cells. F, binding of RBPJ and NICD to the DAB1 promoter. G, RBPJ-dependent expression of Dab1 in mouse intestinal tumors. Data are presented as the mean of three independent experiments with SD, unless otherwise stated. *, P < 0.01; #, P < 0.05. Scale bars, 100 μm.
Apc –/–Aes –/–
(Apc/Aes)
Apc –/–Aes –/–Rbpj –/–
(Apc/Aes/Rbpj )
120
Rbpj target genes
A B
C D
E F G
Drosophila
(Krejcí et al. 2009)
Dab1 (dab)Notch1 (Notch)Hes1&5 (E(spl ))
Mouse
(Li et al. 2012)
DAB1 mRNA qRT-PCR
(LS174T cells)
DAB1 promoter ChlP qPCR
(LS174T cells)
DAB1 mRNA qRT-PCR
(mouse tumor)
98 3 55
Apc/Aes
Apc/Aes/Rbpj
100
80
60
40
20
0
4 1.05
4
3
2
1
0
0.5
0IP: IgG RBPJ NICD Apc Apc/Aes Apc/Aes/Rbpj
3
Rela
tive
DA
B1 m
RN
A leve
ls
Rela
tive
DA
B1 m
RN
A leve
ls
Perc
ent in
put
2
1
0
DAPT
Coated
rDII4
(μg/cm2)0 10.1
–
0 10.1
D. melanogaster
dab
M. musculus
Dab1
–10
–10
–10 –5
–5
–5
TSS
TSS
TSS
0 (kb)
0
0
H. sapiens
DAB1
RBPJ-binding
sequences
High affinity (YGTGRGAA)
Low affinity (RTGRGAR)
Primer pair for ChIP q-PCR
mu
Pro
port
ion (
%)
#
#
*
*
*
* **
*
*sm
Shallow → Deep
Invasion depth of tumors
mp se
affecting tumor size or number ( Fig. 1A and B ). These results
indicate that RBPJ-mediated transcription plays a key role in
colorectal cancer progression in the Apc/Aes mouse.
By comparing RBPJ target genes reported in the mouse
( 6 ) and fruit fl y ( 7 ), we noticed that only three genes were
shared between the two species: Dab1 ( dab in Drosophila ), Notch1
( Notch ), and Hes1/5 ( E(spl) ; Fig. 1C ). In this study, we focused
on Dab1 because it enhances neuronal motility during mouse
brain development ( 5 , 8 ). We excluded NOTCH1 for being
in the direct NOTCH signaling feedback loop, whereas exog-
enously introduced Hes1 caused massive death of Colon26
mouse colorectal cancer cells (data not shown). First, we found
that both human and mouse DAB1/Dab1 genes contain high-
affi nity RBPJ-binding motifs in their proximal promoter regions
as in Drosophila ( Fig. 1D ), suggesting evolutionally conserved
transcription by RBPJ ( 9 ). Consistently, immobilized NOTCH
ligand DLL4 induced DAB1 mRNA in cultured LS174T human
colorectal cancer cells, whereas the γ-secretase inhibitor N-[N-
(3,5-Difl uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester
(DAPT) inhibited this induction by blocking NOTCH receptor
activation ( Fig. 1E ). In addition, RBPJ, as well as the activated
NOTCH receptor fragment NICD, was preferentially bound to
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NOTCH–DAB1–ABL–TRIO–RHO Signaling Promotes Colorectal Cancer Metastasis RESEARCH ARTICLE
the DAB1 gene promoter in chromatin immunoprecipitation
(ChIP) assays, suggesting the roles of the RBPJ transcription
complex in DAB1 expression ( Fig. 1F ). We obtained similar
results with another human colorectal cancer cell line, Colo205,
as well (Supplementary Fig. S1A and S1B). These two cell lines
expressed DAB1 mRNA at higher levels than normal colonic
mucosa, whereas others did at lower levels (Supplementary
Fig. S1C). Consistent with these cultured cell data, we found
RBPJ-dependent induction of Dab1 mRNA and protein in the
intestinal cancer of Apc/Aes mice ( Fig. 1G and Supplementary
Fig. S1D). These results indicate that the DAB1/Dab1 gene is
one of the NOTCH signaling transcription targets of RBPJ in
colorectal cancer cells in both humans and mice.
Importantly, homozygous null mutation of Dab1 in the intes-
tinal epithelium inhibited invasion and intravasation of the
4-HT–dosed Apc +/Δ716 ; Aes Flox/Flox ; Dab1 Flox/Flox ; TgvCre ERT2 mouse
intestinal tumors (abbreviated as Apc −/− Aes −/− Dab1 −/− or Apc/
Aes/Dab1 ). Namely, the tumor intravasation incidence (i.e., the
number of mice that showed at least one lesion of intravasat-
ing tumor into the intestinal wall blood vessels per animal) was
80% (4 of 5) in Apc/Aes mice, whereas it was 0% (0 of 5) for Apc/
Aes/Dab1 mice. Refl ecting this difference, the depth of tumor
invasion was signifi cantly reduced in the Apc/Aes/Dab1 mice as
compared with the Apc/Aes mice ( Fig. 2A and B ). In addition,
constitutive knockdown of the DAB1 gene by shRNA in LS174T
cells signifi cantly inhibited lung metastasis (employed as the
surrogate for liver metastasis; ref. 3 ) after their transplantation
into the nude mouse rectum, without affecting the primary
tumor size ( Fig. 2C–F ). On the other hand, exogenous expres-
sion of DAB1 in RKO, one of the low-expresser human colorectal
cancer cell lines (Supplementary Fig. S1C), increased the metas-
tasis frequency approximately 4 times upon rectal transplanta-
tion, without affecting the primary tumor size or the growth rate
in culture ( Fig. 2G–J and data not shown). Although the primary
tumors of DAB1 high-expresser LS174T cells showed lesions
of intravasation to blood vessels (Supplementary Fig. S2A and
S2B), such histology was not observed in the tumors of sh DAB1 -
expressing LS174T cells (Supplementary Fig. S2C). Collectively,
Figure 2. Reduced expression of DAB1 blocks invasion and metastasis of mouse intestinal tumors, whereas its increase stimulates metastasis. A and B, Dab1 is critical for progression of colorectal cancer in genetically altered mice. Note that after induction of intestinal epithelium–specifi c Cre recombinase–mediated mutagenesis, the tumor genotypes were Apc −/− Aes −/− and Apc −/− Aes −/− Dab1 −/− , respectively. The same methods and keys are employed as in Fig. 1A and B ; n = 5. Boxed areas are magnifi ed in the insets. Arrow in A, intravasating tumor cells inside the vascular lumen (*) with red blood cells. C–F, knockdown of DAB1 inhibits metastasis of colorectal cancer transplants. LS174T cells were transduced with an expression virus encoding EGFP and nonsilencing shRNA (Ns) or shRNA against DAB1 (sh DAB1 ; C). Two clonal lines (#1 and #2) were established for each transductant and injected into the rectal smooth muscle of nude mice. Their lung metastases were evaluated under a fl uorescent dissection microscope (D), and their numbers were scored (E). The primary tumor volumes were not affected by expression of either sh DAB1 (F). G–J, expression of DAB1 stimulates colorec-tal cancer metastasis in vivo . RKO cells were transduced with expression viruses for EGFP and DAB1, or EGFP alone (G), and the established clones were transplanted into the nude mouse rectum. Microscopic examination (H) and quantifi cation (I) were performed as in D and E, respectively. The primary tumor volume was not affected by expression of DAB1 in RKO (J). Data are presented as the mean of three independent experiments with SD, unless otherwise stated. *, P < 0.01. Scale bars, 100 μm (for A) and 500 μm (for D and H).
Apc –/–Aes –/–
(Apc/Aes)
Apc –/–Aes –/–Dab1 –/–
(Apc/Aes/Dab1)
120
100
80
60
40
20
1.4LS174T (high DAB1)
A B
C D E F
G H I J
shDAB1
DAB1
Ns
RKO (low DAB1)
1.21.00.80.60.40.2
0
0
2
4
6
8
10
–
Ns shDAB1
–
DAB1 – DAB1 – DAB1
Rela
tive
DA
B1 m
RN
A leve
lsR
ela
tive
DA
B1 m
RN
A leve
ls
EG
FP
-labele
d R
KO
EG
FP
-labele
d L
S174T
# 1 # 2 # 1 # 2
0
125*
*
100
3
2
1
0
75
50
25
0
300
2.5RKO
2.0
1.5
1.0
0.5
0
200
100
0
mu sm
LS174T
mp se
Pro
port
ion (
%)
Num
ber
of lu
ng m
eta
sta
ses
Num
ber
of lu
ng m
eta
sta
ses
Recta
l tu
mor
volu
me (
cm
3)
Recta
l tu
mor
volu
me (
cm
3)
Apc/Aes
Apc/Aes/Dab1
Invasion depth of tumors
*
**
Shallow → Deep
Ns shDAB1
# 1 # 2 # 1 # 2
Ns shDAB1
# 1 # 2 # 1 # 2
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Sonoshita et al.RESEARCH ARTICLE
these results suggest prometastatic roles for DAB1 in NOTCH
signaling–dependent colorectal cancer progression.
DAB1 Protein Is Tyrosine-Phosphorylated in Colorectal Cancer Cells by ABL Kinase, and Phosphorylated DAB1 Helps ABL Autophosphorylation Reciprocally
DAB1 was identifi ed as one of the proteins that bound
ABL as well as SRC family tyrosine kinases ( 10 ). In RKO
colorectal cancer cells in which the endogenous level of
DAB1 mRNA expression is low (Supplementary Fig. S1C),
its exogenous expression more than doubled the number of
Matrigel-invading cells ( Fig. 3A ). Notably, dasatinib, an ABL/
SRC dual inhibitor, suppressed Matrigel invasion down to 5%
to 10%, either with or without additional expression of DAB1.
However, the SRC family–specifi c inhibitor PP2 had little
effect in the same assay ( Fig. 3A ), suggesting that the effect
of dasatinib was caused mainly by inhibition of ABL. Con-
sistently, we found dose-dependent suppression of Matrigel
invasion by the ABL inhibitor imatinib ( Fig. 3B ). The invasion
inhibition by these inhibitors was not affected substantially
by their viability suppression which was <15% (see Methods).
Figure 3. Tyrosine phosphorylations of both DAB1 and ABL are essential for invasion of colorectal cancer cells. A, exogenous expression of DAB1 mRNA increased Matrigel invasion of RKO cells, and SRC/ABL dual inhibitor dasatinib (Das; at 0.01 μmol/L ) showed a tighter suppressive effect on Matrigel invasion of RKO cells than the SRC inhibitor PP2 (at 10 μmol/L), either with or without additional expression of DAB1. Imatinib (Ima; 10 μmol/L) also showed signifi cant inhibition of invasion (see B below). B, the ABL inhibitor imatinib blocks Matrigel invasion of RKO cells in a dose-dependent man-ner. C, ABL knockdown in human colorectal cancer cells inhibits Matrigel invasion. RKO cells were transfected with either of two independent siRNA sets (#1 or #2), each consisting of distinct siRNAs against ABL1 (si ABL1 -1 and -2) and ABL2 (si ABL2 -1 and -2). Ns, nonsilencing control. D, DAB1 increases ABL Tyr-kinase activity in human colorectal cancer cells. Lanes 1 to 3, HCT116 cells were transfected with the same amount of FLAG-tagged WT DAB1 (lane 2) or its 5YF mutant (lane 3), and their phospho-Tyr (pY) contents were determined by immunoprecipitation–Western blotting (IP-WB). Lanes 4 to 7, HCT116 cells were cotransfected with FLAG-tagged DAB1 (DAB1-FLAG) and HA-tagged WT ABL1b (ABL1b-HA). Note that the input amount of DAB1-FLAG cDNA for lanes 6 and 7 was the same as that for lanes 2 and 3. Lanes 8 to 11, HCT116 cells were cotransfected with the same set of DAB1 cDNA as in Lanes 4 to 7, and KD mutant of ABL1b. E, ABL is found in DAB1 immunoprecipitation (IP) of intestinal tumors in Apc/Aes mice as analyzed by Western blotting (left), whereas DAB1 is found in ABL IP, reciprocally (right). WB indicates antibodies used for detection of ABL or DAB1 by Western blotting. Data are presented as the mean of three independent experiments, with SD. *, P < 0.01.
Matrigel invasion assay (RKO cells)A B
DC
E
Matrigel invasion assay (RKO cells)
qRT-PCR
Oligo set # 1 # 2
Matrigel invasion assay (RKO cells)
150
100
50
0
ABL1b-HA
HAIP: HA
IP: lgG lgG
anti-
DAB1
anti-
ABL
DAB1-FLAG
IP: FLAGFLAG
0
WT WT WT
WT
– – –
Imatinib [ABL inh.] (μmol/L)0.1 101
*
*
**
**
*
**
Num
ber
of cells
inva
ded
(per
×400 fie
ld)
Num
ber
of cells
inva
ded
(per
×200 fie
ld)
Rela
tive
mR
NA
expre
ssio
n leve
ls
Num
ber
of cells
inva
ded
(per
×200 fie
ld)
200
150
100
50
0
150
100
50
01.2
1.0
0.8
0.6
0.4
0.2
0
Ns1
Ns2
Both
Both
Both
siA
BL1
siA
BL1
siA
BL2
siA
BL2
– DAB1
– PP2 Das Ima – PP2 Das Ima
ABL1
ABL2
5YF 5YF 5YF WB:
WB:
ABL
DAB1
1 2 3 4 5 6 7 8 9 10 11
pY
pY
KD
Apc –/–Aes –/– (Apc/Aes) tumors
– – –
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NOTCH–DAB1–ABL–TRIO–RHO Signaling Promotes Colorectal Cancer Metastasis RESEARCH ARTICLE
The ABL subfamily of tyrosine kinases is conserved evolu-
tionarily, consisting of ABL1 and ABL2 (aka ARG) in mam-
mals, and has pleiotropic roles in cell proliferation, migration,
etc. ( 11 ). Knockdown of the ABL1 and ABL2 genes ( Fig. 3C ,
bottom) in RKO cells reduced the mRNA levels below 40%,
and Matrigel invasion to 15% to 20%, without affecting cell
viability ( Fig. 3C , bottom and top, respectively; see also Meth-
ods). These results show that ABL tyrosine kinase activity is
essential for Matrigel invasion of human colorectal cancer
cells, implicating its role in colorectal cancer progression.
It is known that ABL phosphorylates itself for maximal
kinase activity ( 12 ). In neurons and HEK293 cells, DAB1
enhances the kinase activity of FYN ( 13 ). In an analogy, we
found that simultaneous expression of DAB1 and ABL1b, the
major splice variant of ABL1 ( 11, 12 ), increased the level of
Tyr-phosphorylated ABL1b (pY-ABL1b) in a dose-dependent
manner in RKO cells ( Fig. 3D ; compare lanes 4–6). For
neuronal migration, Tyr-phosphorylated DAB1 (pY-DAB1)
is essential, because it is compromised by expression of the
“5YF” DAB1 mutant, in which fi ve Tyr residues are replaced
with Phe, a structurally similar but nonphosphorylatable aa
( 8 ). In the RKO cells expressing 5YF-DAB1, we found that the
pY-ABL1b level remained low ( Fig. 3D ; lane 7), suggesting
that ABL Tyr phosphorylation was dependent on pY-DAB1.
Consistently, wild-type (WT) DAB1 was Tyr-phosphorylated
in the presence of WT ABL1b, but not a kinase-dead (KD)
mutant ABL1b(K290R) ( Fig. 3D ; lanes 9 and 10; ref. 14 ).
Consistent with a previous report ( 10 ), ABL coprecipitated
with DAB1 with anti-DAB1 Ab from the lysate of Apc/Aes
mouse intestinal tumors, whereas DAB1 coprecipitated with
ABL with anti-ABL Ab in a reciprocal experiment ( Fig. 3E ),
suggesting their physical interaction in vivo either directly
or through additional proteins. Importantly, imatinib sig-
nifi cantly inhibited colorectal cancer invasion in Apc/Aes mice
without affecting the tumor size or number ( Fig. 4A–C ). Con-
sistently, the tumor intravasation incidence was decreased
from 100% (5 of 5) in vehicle-treated Apc/Aes mice to 0% (0 of
5) in imatinib-dosed mice. These results indicate that DAB1 is
phosphorylated when coexpressed with ABL in colorectal can-
cer cell lines, and, reciprocally, pY-DAB1 stimulates ABL auto-
phosphorylation, which suggests in vivo activation to promote
colorectal cancer invasion as one of the downstream effects of
NOTCH signaling.
One of the Targets Phosphorylated by Activated ABL in Colorectal Cancer Cells Is the RAC/RHOGEF Protein TRIO
In an attempt to determine the substrate(s) of ABL respon-
sible for colorectal cancer cell invasion, we looked into Dro-
sophila biology. As a downstream effecter of Drosophila ABL
(dABL), Triple functional domain (dTRIO) plays key roles in
fruit fl y neuronal migration ( 15 ). TRIO belongs to the DBL
family of guanine nucleotide exchange factor (GEF) proteins
and can activate the RHO family of small GTPases, well-
characterized and essential regulators of cell motility ( 16,
17 ). TRIO carries two GEF domains: one for RAC (GEF1) and
the other for RHO (GEF2; ref. 18 ; Fig. 5C ; top). Therefore, we
speculated that RAC and/or RHO was also activated by TRIO
in human colorectal cancer cells to promote their invasion.
Interestingly, RHO was activated signifi cantly as the GTP-
bound form in invasive ( Apc/Aes ) mouse colon cancer compared
with benign ( Apc ) adenomas, whereas RAC-GTP was below the
detection level in Apc/Aes tumors, although it showed a weak
band in Apc adenomas ( Fig. 5A ) and a strong band in a control
experiment (Supplementary Fig. S3A). Consistently, inhibition
of RHO by C3T ( 19 ), or its immediate downstream effector
ROCK by Y-27632 ( 20 ), blocked the Matrigel invasion and
TEM of RKO human colorectal cancer cells in a dose-depend-
ent manner ( Fig. 5B and Supplementary Fig. S3B, respectively).
This inhibition of invasion appears to be mostly due to direct
effects, although a small fraction may be attributable to inhibi-
tion of viability (up to 20% at higher doses; see Methods). On
the other hand, RAC inhibition by NSC23766 or RAC Inhibi-
tor II ( 21 ) did not affect Matrigel invasion (Supplementary
Fig. S3C and S3D). These results suggest that the NOTCH
signaling–dependent Matrigel invasion and TEM of colorectal
cancer cells in culture ( 3 ) are mediated by RHO activation.
Furthermore, knockdown of TRIO in HCT116 cells (Supple-
mentary Fig. S3E and S3F) reduced their Matrigel invasion
( Fig. 5D ) and lung metastasis rate upon rectal transplantation
( Fig. 5E ) without affecting the primary tumor size or growth
rate in culture (Supplementary Fig. S3G, and data not shown),
suggesting that TRIO RHOGEF activity plays a key role in the
invasion of NOTCH receptor–activated colorectal cancer cells.
In Colorectal Cancer Cells, TRIO Y2681 Is One of the Major Phosphorylation Sites Activated by DAB1–ABL
In Drosophila S2 cells, dTRIO is Tyr-phosphorylated in the
presence of dABL ( 15 ). Also, in HCT116 colorectal cancer
cells, we found that some Tyr residues of TRIO were heavily
Figure 4. ABL kinase inhibitor imatinib can block invasion of mouse intestinal tumors. A and B, imatinib suppresses invasion of endogenous colorectal cancer in Apc/Aes compound mutant mice ( n = 5 mice for each group). The same methods and keys were employed as in Fig. 1A and B . C, the intestinal tumor number or size was not affected by imatinib in Apc/Aes mice. Scale bars, 100 μm. Error bars, SD. *, P < 0.01.
Apc –/–Aes –/– (Apc/Aes) tumors
VehicleA
B C
Vehicle
120
300
200
100
0
100
80
60
40
*
*
*
Pro
port
ion
(%)
Tum
or n
umbe
r
20
0mu sm mp se
φ < 0.5
0.5 ≤ φ < 2 2 ≤ φ
Invasion depth ofApc/Aes tumors
Apc/Aes tumor size
Imatinib
ImatinibVehicleImatinib
Tumor diameter (mm)Shallow → Deep
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Sonoshita et al.RESEARCH ARTICLE
Figure 5. TRIO GEF protein is phosphorylated at Y2681 by ABL, and activates RHO, leading to colorectal cancer invasion. A, activation of RHO, but not RAC, in the invading intestinal tumors of Apc/Aes mice. In RHO-GTP and RAC-GTP pull-down assays, tissues of normal intestinal mucosa (N) and tumors (T) were analyzed from Apc control, and Apc/Aes mutant mice. The amounts of total RHO and RAC proteins are also shown. Representative results of three independent pairs. B, inhibition of RHO or ROCK suppresses Matrigel invasion of colorectal cancer cells in culture. RKO cells were assayed in the presence of the RHO inhibitor C3T or ROCK inhibitor Y-27632 at two doses, respectively ( n = 5). C, schematic drawing of TRIO domain structure inspired by earlier work from Debant et al. ( 18 ) and phosphorylatable Tyr residues. Shown on top are domains of the WT human TRIO (TRIO WT) that contains 3097 aa. Rectangles show specifi c domains as indicated. GEF, guanine nucleotide exchange factor domain; Ig, immunoglobulin-like domain; SH3, SRC homology 3 domain; STK, serine-threonine kinase domain. Below the TRIO WT structure, the positions of Tyr (Y) to Phe (F) mutations are shown as red “F”s. D, knockdown of TRIO suppresses colorectal cancer cell invasion. Two independent siRNAs were used for nonsilencing (Ns) and TRIO -silencing (si TRIO ), respectively. –, untransfected control. E, knockdown of TRIO suppresses colorectal cancer metastasis. TRIO -knockdown (sh TRIO ) and control (Ns) HCT116 cells were established and transplanted into the rectal mucosa of nude mice. Six weeks later, their lung metastatic foci were scored ( n = 5 mice for each group). (continued on following page)
RHO- or RAC-GTP
pull-down
(Mouse intestine)
Matrigel invasion assay (RKO)
Matrigel invasion assay
(HCT116)Rectum −> Lung (HCT116)
***
**
*200
150
100
50
0
200
150
100
50
0–
600
500
400
300
200
100
01 3
C3T(μg/mL)
Y-27632(μmol/L)
101–
Apc
N NT TRHO-GTP
A B D E
C
RAC-GTP
Total RHO
Total RAC
Y75
Spectrin repeats
TRIO WT
18YFs
7YFs-a
7YFs-b
11YFs-b
4YFs
Y1990F (0.941)
Y2562F (0.516)
Y2681F (0.649)
Y2757F (0.961)
(pY probability)
GEF1 GEF2SH3 SH3 lg STK
Y367
Y395
Y491
Y699
Y1140
Y1161
Y1307
Y1362
Y1984
Y1990
Y2117
Y2286
Y2562
Y2659
Y2681
Y2732
Y2757
Y2796
Apc/Aes
Nu
mb
er
of
ce
lls inva
de
d
(pe
r ×
20
0 f
ield
)
Nu
mb
er
of
ce
lls inva
de
d
(pe
r ×
20
0 f
ield
)
Nu
mb
er
of
lun
g m
eta
sta
se
s
# 1 # 2 # 1 # 2 # 1 # 2 # 1 # 2
Ns siTRIO Ns shTRIO
phosphorylated when simultaneously expressed with DAB1
and ABL1b ( Fig. 5F , lane 4), but not with DAB1 5YF mutant
(lane 5) nor with ABL1b KD mutant (lane 7). These results
suggested that Tyr-phosphorylation of TRIO was dependent
on ABL kinase activity.
To determine the particular phospho-Tyr residues in
TRIO, we next screened its primary amino acid sequence with
NetPhos2.0 software ( 22 ). It predicted the probability of phos-
phorylation for all 61 Tyr residues in TRIO, and identifi ed 30
as likely candidates with scores above the threshold of 0.5.
Among a series of TRIO tyrosine-phenylalanine (YF) mutants
( Fig. 5C ), we found signifi cant reduction in the pY level of
TRIO 18YF compared with TRIO WT when expressed in RKO
cells ( Fig. 5G , lanes 1 and 2). Although the colorectal cancer
cells expressing TRIO 7YFs-a or TRIO 7YFs-b contained pY-
TRIO at a similar level to control TRIO WT, those expressing
TRIO 11YFs-b that carried an additional four YF mutations at
residues 1990, 2562, 2681, and 2757 had a markedly reduced
level of pY-TRIO ( Fig. 5G , lane 5). As anticipated, the pY
level was similarly reduced in cells expressing the TRIO 4YFs
mutant in which only the additional four tyrosine residues
were mutated ( Fig. 5G , lane 6). Among these four Tyr residues,
we identifi ed Y1990 and Y2681 as the key targets of phospho-
rylation by DAB1–ABL, because expression of either Y1990F
or Y2681F mutant (Fig. 5H, lanes 2 and 4), but not Y2562F
or Y2757F (lanes 3 and 5), markedly reduced the pY levels in
the colorectal cancer cells. Notably, the aa sequence adjoin-
ing TRIO Y2681 has a strong similarity to the ABL target site
consensus ( 11 ). It is also known that dTRIO protein binds to
dABL ( 15 ). Accordingly, it is likely that ABL directly phospho-
rylates TRIO Y2681 in colorectal cancer cells.
To determine the direct effects of introduced proteins, we
employed the TetON system to conditionally express TRIO,
together with DAB1 and ABL1b, and confi rmed that TRIO
Y2681F failed to promote Matrigel invasion of RKO cells,
although TRIO WT and TRIO Y1990F could do so ( Fig. 5I ).
Because induction of TRIO WT alone in RKO cells did not
increase Matrigel invasion (Supplementary Fig. S3H), these
results indicate that TRIO is the effector of DAB1–ABL-
induced invasion. In RHOGEF GTP-exchange assays per-
formed in vitro using purifi ed proteins, TRIO WT enhanced the
exchange of GDP in RHOA-GDP with GTP (i.e., activation to
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NOTCH–DAB1–ABL–TRIO–RHO Signaling Promotes Colorectal Cancer Metastasis RESEARCH ARTICLE
F
I J K
G HIP-WB
IP-WB IP-WB– + + + + + + +
++
++
++
++
++
++
++
++
++
++
++
++
–– –
–– –
T7-TRIO
T7-TRIO –
– – ++– +
T7
T7-TRIO T7-TRIOABL1b-HA
DAB1-FLAGABL1b-HA
DAB1-FLAGWB:
pY
1 2 3 4 5 6 1 2 3 4 5 6
IP: T7
IP: T7 IP: T7T7
WB:
pY
T7IP: HA
1,400
*
*
*
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
T7-TRIO
WT WT[ima] Y2681F
Cell-free RHOGEF assayCell-free RHOGEF assay
RHOA+TRIO
RHOA
1,300
1,200
1,100
1,0000 1 2 3
Time (min)Induction by Dox
WT Y2681F InducedcDNAs
4 5 6 7 8 9 10
Num
ber
of cells
inva
ded
(per
×200 fie
ld)
Rela
tive
lum
inescence
RH
O a
ctiva
tion (
fold
)
100
80
60
40
20
0
Matrigel invasion assay (RKO TetON)
21 3 4 5 6 7 8
HA
ABL1b-HADAB1-FLAG
IP: FLAGFLAG
WB:
WB:
pY
T7
HA
ABL1b-HA/DAB1-FLAG
FLAG
pY
pY
KD
WT WT WT
18YFs
7YFs-a
7YFs-b
11YFs-b
4YFs
WT
Y1990F
Y2562F
Y2681F
Y2757F
4YFs5YF 5YF
WT
Figure 5. (Continued) F, TRIO is Tyr-phosphorylated in the presence of ABL and DAB1 in colorectal cancer cells. HCT116 cells were transduced with or without an expression construct for T7-tagged TRIO (T7-TRIO), cotransduced with or without HA-tagged ABL1b (ABL1b-HA), and/or FLAG-tagged DAB1 (DAB1-FLAG). Phospho-Tyr (pY) contents were determined by Western blotting (WB) after immunoprecipitation (IP). For ABL1b, either WT or KD mutant was used. G, some tyrosine residues in the C-terminal half of TRIO are phosphorylated by ABL. WT or YF mutants of T7-TRIO were expressed simultane-ously with ABL1b-HA and DAB1-FLAG, followed by Western blot analysis of pYs. H, Y1990 and Y2681 in TRIO are phosphorylated in the presence of ABL and DAB1. I, stimulation of colorectal cancer cell invasion in Matrigel by expressing TRIO WT, but not by TRIO Y2681F. Clonal RKO TetON derivatives were constructed to express ABL1b-HA/DAB1-FLAG simultaneously with T7-TRIO WT or T7-TRIO Y2681F in a doxycycline (Dox)-inducible manner, and assayed for protein expression and Matrigel invasion. J, cell-free RHOGEF GTP exchange assay. A representative result of three experiments is shown. K, TRIO pY2681 is critical for the RHOGEF activity. T7-tagged TRIO (WT or Y2681F) was purifi ed, and its RHOGEF activity for 10 minutes was determined as shown in J. WT[ima], WT TRIO protein purifi ed from imatinib (10 μmol/L)-treated HEK293T cells. All quantitative data are presented as the mean with SD. *, P < 0.01 in three independent experiments unless otherwise stated.
RHOA-GTP), as reported (ref. 18 ; Fig. 5J ). Importantly, TRIO
WT protein purifi ed from imatinib-treated transductant
cells and TRIO Y2681F mutant protein showed signifi cantly
decreased RHOGEF activity (40%–50%; Fig. 5K ) without
affecting RACGEF activity (Supplementary Fig. S3I), sug-
gesting that phosphorylation at Y2681 by ABL is essential for
its maximal RHO activation. Together, these results indicate
that ABL causes phosphorylation of TRIO at Y2681 and
increases the level of RHO-GTP, stimulating invasion of colo-
rectal cancer cells in culture.
TRIO pY2681 Signifi cantly Correlates with Poor Prognosis of Patients with Colorectal Cancer
To determine the clinical relevance of the TRIO phospho-
rylation in colorectal cancer, we raised polyclonal antibod-
ies specifi c for TRIO pY1990 and TRIO pY2681 peptides.
Namely, antibodies were raised against phospho-Tyr–
containing peptides surrounding Y1990 and Y2681, respec-
tively, and absorbed with the same sequence peptides lacking
Tyr-phosphorylation. We fi rst confi rmed their specifi cities
using HEK293T cells that expressed TRIO WT, Y1990F, or
Y2681F mutant (Supplementary Fig. S4A and S4B). We also
tested them on human colorectal cancer cell line mouse
xenograft tumors by immunohistochemistry (IHC). The anti-
TRIO pY2681 cytoplasmic staining disappeared upon expres-
sion of sh DAB1 or sh TRIO (Supplementary Figs. S2B and
S2C; S4C and S4D), verifying their specifi cities.
We then analyzed surgical specimens of colorectal cancer
by IHC with these antibodies. Consistent with the above
results that TRIO pY1990 hardly affected Matrigel invasion
of RKO cells, the disease-specifi c survival rates (DSS) were
similar between the TRIO pY1990-low and -high patients
( P = 0.9, n = 129; data not shown). In contrast, the patients
with high levels of TRIO pY2681 in their primary colorectal
cancer showed a statistically signifi cant reduction in their
DSS, as compared with those who had TRIO pY2681-low
colorectal cancer (all stages included, n = 337, P < 0.001 in the
log-rank test; Fig. 6A , left). When the stage II colorectal can-
cer subpopulation was examined, the 5-year DSS was 100%
for TRIO pY2681-low patients, whereas it was approximately
80% for the TRIO pY2681-high patients ( n = 115, P = 0.015;
Fig. 6A , center). Even within the stage IV subpopulation,
the 5-year DSS was approximately 50% for TRIO pY2681-
low patients, in contrast to only approximately 10% for the
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Sonoshita et al.RESEARCH ARTICLE
Figure 6. Phosphorylation of TRIO Y2681 in human colorectal cancer (CRC) is correlated with poorer prognosis. A, (left) patients of all stages were combined; 150 cases were TRIO pY2681-low, whereas 187 cases were TRIO pY2681-high ( P < 0.001 in log-rank test). Center, patients of stages II; the TRIO pY2681-low group ( n = 45) and the TRIO pY2681-high group ( n = 70, P = 0.015). Right, stage IV patients; the TRIO pY2681-low patients ( n = 11) and TRIO pY2681-high patients ( n = 46, P = 0.006). B, cytoplasmic staining for TRIO pY2681 in colorectal cancer cells (arrowheads). Compare with the adjacent normal mucosa (N) or lymphoid follicle (L). Boxed area is magnifi ed in the inset. C–E, staining for TRIO pY2681 is found in colorectal cancer cells invading into the stroma (arrowheads). F–I, immunohistochemical analysis of colorectal cancer sections for stem cell and EMT markers in relation to TRIO pY2681 staining. Stem cell markers LGR5 (F) and SOX9 (G), as well as cytokeratin 18 (CK18; H), were stained in the same series of sections as that stained for TRIO pY2681 (I). Similar lesions were selected and photographed at the same magnifi cation. Note that the staining for CK18 at the invading front is weakened (arrows). J, correlation between NICD and TRIO pY2681 immunostaining in human colorectal cancer. Shown is a representative set of adjoining serial sections (left and right). Arrows indicate positive staining whereas arrowheads indicate negative staining. Insets show higher magnifi ca-tion of boxed areas. Hematoxylin counterstaining. Scale bars, 100 μm for B and C, 20 μm for F to I, and 50 μm for J.
Stages 0–IVA
B C
D E
F G J
H I
LGR5 SOX9
CK18 TRIO pY2681
100
80Low
High
P < 0.001
P = 0.006
P = 0.015
High
High
Low
Low
60
40
20
01
NICD
NIC
DH
igh
TR
IO p
Y2
68
1H
igh
NIC
DLow
TR
IO p
Y2
68
1Low
TRIO pY2681
2 3 4Su
rviv
al o
f C
RC
pa
tie
nts
(%
)
5 6 7 8
100
80
60
40
20
01 2 3
(Years)4 5 6 7 8
100
80
60
40
20
01 2 3
Invasion direction
4 5 6 7 8
Stage II Stage IV
TRIO pY2681-high patients ( n = 57, P = 0.006; Fig. 6A , right).
Accordingly, the phosphorylation status of TRIO Y2681 may
help future prognostic studies, together with the currently
available biomarkers.
In the TRIO pY2681-high specimens, we found strong
immunoreactivity in the cytoplasm of colorectal cancer cells
compared with the adjacent normal mucosa ( Fig. 6B ). Nota-
bly, TRIO pY2681 staining was found also in the isolated,
budding, or invading colorectal cancer cells in the stroma
(arrowheads in Fig. 6C–E ), supporting the thesis that TRIO
pY2681 is critical for human colorectal cancer invasion.
Interestingly, these budding colorectal cancer cells expressed
some stem cell markers such as LGR5 ( 23 ) and SOX9 ( 24 )
and showed signs of partial epithelial–mesenchymal transi-
tion (EMT), such as decreased expression of cytokeratin
18 and E-cadherin, consistent with earlier reports (ref. 25 ;
Fig. 6F–I , and Supplementary Fig. S4E). Notably, Colon26
mouse colorectal cancer cells showed reduced levels of
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NOTCH–DAB1–ABL–TRIO–RHO Signaling Promotes Colorectal Cancer Metastasis RESEARCH ARTICLE
the EMT-related transcription factors SLUG and TWIST (but
not SNAIL) when AES expression was induced and NOTCH
signaling was inhibited (Supplementary Fig. S4F). These
results show that TRIO pY2681 and partial EMT can be
found at the invasion front of colorectal cancer where signs of
increased stemness are detected. They suggest that activation
of TRIO RHOGEF may play a role in colorectal cancer stem
cell functions associated with partial EMT. Because EMT-
related molecular changes are induced even in adenomas of
Min mice (i.e., in an early stage of tumorigenesis; ref. 26 ),
however, the relationship of NOTCH signaling with stemness
about TRIO pY2681 will require further study.
To further confi rm that TRIO phosphorylation at Y2681
was caused by upstream activation of NOTCH signaling in
human colorectal cancer, we immunostained the cleaved and
activated NOTCH receptor NICD and TRIO pY2681 in serial
pathologic sections. The immunostaining of nuclear NICD
positively correlated with that of cytoplasmic TRIO pY2681
( n = 40, P < 0.01 in χ 2 tests; Fig. 6J ). Consistent with these human
clinical data, we found a much higher pY2681 content in the
TRIO protein immunoprecipitated from the Apc/Aes mouse
intestinal cancer than that from the Apc adenoma (Supple-
mentary Fig. S5A). These results support our interpretation
that TRIO pY2681 is caused by activation of NOTCH signal-
ing in both human and mouse colorectal cancers, although
pathways other than DAB1–ABL phosphorylation may also
induce TRIO pY2681.
Bound with TLE1, AES suppresses colorectal cancer pro-
gression by inhibiting NOTCH signaling ( 3 ). Notably, nuclear
expression of AES was inversely correlated with TRIO pY2681
levels in human colorectal cancer specimens as determined by
IHC ( n = 102, P < 0.01; representative photographs in Supple-
mentary Fig. S5B). Furthermore, exogenous expression of AES
and TLE1 inhibited ABL activity as determined by Tyr-phos-
phorylation of DAB1 in RKO cells (Supplementary Fig. S5C).
We have already shown that immobilized NOTCH ligand
DLL4 activates NOTCH signaling in colorectal cancer cells
and promotes their migration, which is suppressed by AES
( 3 ). As anticipated, expression of AES in mouse Colon26
cancer cells reduced the level of RHO-GTP after scratch-
induced migration (see Methods) in a DLL4-coated dish
(Supplementary Fig. S5D). These results collectively indicate
that suppression of NOTCH signaling-induced transcription
inhibits TRIO Tyr-phosphorylation, resulting in a decreased
RHO-GTP level, which reduces invasion and metastasis of
colorectal cancer cells ( Fig. 7 ; see Discussion).
DISCUSSION In the cancer invasion–metastasis cascade ( 1 ), events such as
local invasion, intravasation, and extravasation involve active
cell motility. Tumor cells move either as individual cells or en
masse via collective migration ( 27 ). Individual tumor cells have
two different modes of movement: mesenchymal mode and
amoeboid mode ( 28 ). Although the former is characterized by
an elongated morphology that requires extracellular proteoly-
sis localized at cellular protrusions, the latter is independent
of proteases, and cells have rounded morphology with no
obvious polarity ( 29 ). Members of the RHO family of GTPases
are key regulators of cell movement through their actions on
actin assembly, actomyosin contractility, and microtubules
( 30 ). The amoeboid and mesenchymal modes of movement
are distinguished by their different usage of signaling path-
ways. Namely, the amoeboid mode involves signaling through
RHOA–ROCK, whereas the mesenchymal mode requires extra-
cellular proteolysis ( 31 ) for RAC-dependent actin protrusions
to be pushed through channels in the extracellular matrix ( 28 ).
Notably, these two modes of motility are observed in invading
tumor cells in vivo ( 29 ), and tumor cells may switch between
the elongated and rounded modes of movement in an adap-
tive response to the microenvironment ( 28 , 31 ). Importantly,
RAC and RHO activities are mutually antagonistic; active RAC
represses RHO activity and vice versa ( 29, 30 ). Although the
ability to switch between modes of movement gives cells adapt-
ability to a variety of conditions, some tumor cells appear to be
hardwired to stick with a particular mode ( 32 ).
In contrast to single-cell migration, the mechanics of collec-
tive cell migration has been investigated only recently, and the
roles of RHOA and RAC1 have been explored in more collec-
tive processes such as tumor invasion ( 33 ). For example, RHO,
rather than RAC, plays the key role in collective migration
of epithelial cells ( 34 ), by which colorectal cancer very often
invades into the stroma ( 3 ). Consistently, our present results
show that TRIO Y2681F mutation reduces its RHOGEF activ-
ity ( Fig. 5K ) without affecting RACGEF activity (Supplemen-
tary Fig. S3I). Some colorectal cancer cell lines, including
LS174T, appear to migrate by the amoeboid migration mode
Figure 7. A schematic representation of NOTCH signaling through DAB1–ABL–TRIO–RHO that stimulates colorectal cancer invasion and metastasis. Stromal cells, including endothelial cells (EC), can activate NOTCH signaling in colorectal cancer cells and upregulate DAB1 transcrip-tion, activating ABL when the AES level is low. Activated by DAB1, ABL enhances the RHOGEF activity of TRIO by phosphorylating Y2681. Thus, TRIO pY2681 is critical for the RHO activation that contributes to invasion, intra- and extravasation, and metastasis of colorectal cancer cells. In addi-tion, a possibility remains that other products of RBPJ transcription as well as other tyrosine kinases may also be involved in colorectal cancer invasion and metastasis. These results provide a possible prognostic marker in TRIO pY2681 and a therapeutic strategy targeting ABL (e.g., using imatinib and related drugs) to prevent colorectal cancer metastasis.
EC (stromal cell) NOTCH ligand
NOTCH receptor activation (S3 cleavage)
NICDColorectal cancer
cellNucleus
imatinib
RBPJ
DAB1
mRNA
?
?
ABL DAB1
pABL-pDAB1
RHO-GTP
EC
Migration, invasion,
intra- and extra-vasation,
and metastasis
: Possible prognostic markerTRIO pY2681
AES
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Sonoshita et al.RESEARCH ARTICLE
in three-dimensional (3D) Matrigel, whereas others migrate
in an elongated morphology ( 31 ). Yet, these cells can switch
among these migration modes. In the two-dimensional (2D)
scratch assay, colorectal cancer cells migrate as collectives ( 35 ).
Our results above have been obtained using invasive mouse
tumors in vivo , as well as cultured human colorectal cancer cells
in 3D Matrigel invasion and 2D scratch assays. Accordingly,
the data may indicate that invasive mouse tumors and human
colorectal cancer cells in culture share common mechanisms in
terms of utilizing the RHO GTPases.
In the intestinal crypt, NOTCH signaling has been impli-
cated in tissue stem cell functions. For example, activation of
NOTCH signaling in the intestinal epithelium causes expan-
sion of the proliferating cell population ( 36 ), whereas its sup-
pression by a γ-secretase inhibitor reduces proliferation and
causes goblet cell metaplasia ( 37 ). NOTCH signaling is cru-
cial for the maintenance of intestinal crypt bottom columnar
stem cells ( 38 ). However, the downstream molecules directly
involved in stem cell homeostasis remain to be investigated.
At the same time, EMT can cause stem cell–like changes in
breast cancer ( 25 ), although in colorectal cancer, some molec-
ular changes of EMT are detected even in adenomas that lack
overt EMT phenotypes ( 26 ). We have found that the budding
colorectal cancer cells at the invasion front often show TRIO
pY2681, and simultaneously express some stem cell markers,
such as LGR5 and SOX9, with partial signs of EMT ( Fig. 6F–I
and Supplementary Fig. S4E). Accordingly, it is conceivable
that TRIO pY2681 induced by NOTCH signaling activation
is related to the stem cell–like functions of colorectal cancer,
although further investigations are needed.
The above results that imatinib can block DAB1–ABL phos-
phorylation, and the subsequent activation of TRIO-RHO and
invasive motility of colorectal cancer cells, suggest the possibility
that ABL tyrosine kinase inhibitors, such as imatinib and dasat-
inib, may be useful for metastasis prevention as neoadjuvant
chemotherapy and/or post-operative chemotherapy. Notably,
these inhibitors have been studied and proposed for colorec-
tal cancer chemotherapy because of their additional activities
against platelet-derived growth factor receptor ( 39, 40 ) and SRC
( 41 ). The present results can add a novel mechanistic rationale
for the use of these inhibitors against colorectal cancer.
Collectively, we have revealed a novel signaling mechanism
downstream of NOTCH that stimulates colorectal cancer pro-
gression, suggesting the following scenario: Loss of AES activates
NOTCH signaling transcription, and as one of the downstream
effector pathways, induces DAB1 expression, which potenti-
ates ABL autophosphorylation and activation, causing phos-
phorylation of TRIO Y2681 to promote colorectal cancer cell
invasion and TEM through its RHOGEF activity ( Fig. 7 ). We
propose that TRIO pY2681 may be a useful prognostic marker,
and that the ABL activity and its downstream effectors, such
as TRIO, RHO, and ROCK, may be novel therapeutic targets
against malignant progression of colorectal cancer.
METHODS Mutant Mice, Colorectal Cancer Cells, and Transplantation–Metastasis Model
Apc +/Δ716 mutant mice were generated and maintained as described
previously ( 42 ). Apc/Aes mice were constructed and induced for Cre
activity according to previous reports ( 3 , 43 ). To derive triple-mutant
mice, we crossed the Apc/Aes double-mutant mice with Rbpj -null ( 44 )
or Dab1 -null mice (H. Imai and colleagues, unpublished data). For
ABL inhibition in vivo , Apc/Aes mice were treated with 50 mg/kg/day
(ip) of imatinib (LC Laboratories; ref. 45 ) for 9 weeks from 3 weeks of
age. At 12 weeks of age, a total of approximately 100 intestinal tumors
(2 mm < diameter) from 5 mice were scored for the invasion depth in
each mutant genotype or treatment.
We have confi rmed the identity of each colorectal cancer line
(ATCC) recently (July–August 2014) by short tandem repeat analysis
(Takara Bio CDM Center). Colorectal cancer transplantation experi-
ments were performed as described previously ( 3 ). All animal experi-
ments were conducted according to the protocol approved by the
Animal Care and Use Committee of Kyoto University.
Matrigel Invasion and TEM Assays Matrigel invasion and TEM assays were performed as described
previously ( 3 ). Colorectal cancer cells were treated with C3T (ref. 19 ;
Cytoskeleton), Y-27632 (ref. 20 ; Wako), NSC23766, Rac Inhibitor II
( 21 ), or PP2 (ref. 46 ; Calbiochem), imatinib ( 47 ), or dasatinib (ref.
48 ; LC Laboratories) for 17 hours during the assays, after preincu-
bation (for 6 hours for PP2, dasatinib, and imatinib; or 2 hours for
RHO and ROCK inhibitors) and passage with drug concentrations
used in the original reports above. Although up to approximately
15% survival inhibition was found in the MTS assay by dasatinib
and imatinib, and up to 15% and 20% by C3T and Y-27632, respec-
tively, they did not affect the interpretation of the invasion assays
substantially.
Knockdown/Overexpression Experiments Forty-eight hours after transfection of specifi c siRNA oligonucle-
otides (QIAGEN), colorectal cancer cells were subjected to Matrigel
invasion, gene expression, or MTS assays in which we did not
fi nd any signifi cant changes in cell viabilities between transfec-
tion groups. To establish stable knockdown cells for transplanta-
tion experiments, we prepared lentiviral particles using sequences
sh DAB1 #1 (AAGGATTAAGTAGGATGTCAA) and sh DAB1 #2 (CCG
GTACAAAGCCAAATTGAT) or sh TRIO #1 (CCGGGAATGTATGGA
TACGTA) and sh TRIO #2 (CAACGGAGAGTCCATGTTAAA), and
pLB lentiviral vector (Addgene).
WT mouse Dab1 cDNA ( 8 ) was inserted into retroviral vector pMX-
IRES-EGFP ( 49 ). Viral particles were prepared and infected to RKO
cells, and EGFP + cells were confi rmed for stable Dab1 expression.
dab/Dab1/DAB1 Promoter Analysis Fruit fl y dab , mouse Dab1 , and human DAB1 gene promoters were
extracted from the UCSC Genome Browser Database and determined
for high- and low-affi nity Su(H)/RBPJ-binding sites, as described
previously ( 50 ).
rDLL4 Experiments Culture plate surfaces were coated with recombinant DLL4 ligand
(rDLL4; R&D) at 4°C overnight, and then washed once with PBS
and seeded with colorectal cancer cells ( 3 ). Four hours after plating,
mRNA was extracted and analyzed for DAB1 mRNA by qRT-PCR.
qPCR TaqMan primers/probes were purchased from ABI. ChIP analy-
ses were performed on colorectal cancer cell lysates using anti-
RBPJ (Institute of Immunology) and anti-NICD (Cell Signaling
Technology) antibodies and hDAB1ChIP.F2 (CAAGCTCTGTGCTT-
GTCTCA) and hDAB1ChIP.R2 (GTAGCTGTGTGGTCTTATCA)
primers ( Fig. 1D , paired triangles) according to a published protocol
( 51 ).
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FEBRUARY 2015�CANCER DISCOVERY | 209
NOTCH–DAB1–ABL–TRIO–RHO Signaling Promotes Colorectal Cancer Metastasis RESEARCH ARTICLE
IHC Frozen and paraffi n-embedded tissues of mice were prepared
according to the standard procedures. Human colorectal cancer tis-
sues ( n = 337, stages 0–IV) had been resected from patients in Kyoto
University Hospital who had undergone operations with informed
consent between 2005 and 2008, with the protocol approved by the
Ethics Committee of Kyoto University. Sections were stained with
hematoxylin and eosin or for DAB1 (SIGMA), AES ( 3 ), NICD (Cell
Signaling Technology), TRIO pY1990, or TRIO pY2681 (prepared as
described below). For evaluation of IHC on clinical specimens, 2 to 3
examiners scored independently, followed by a consensus discussion
with advice from a board-certifi ed clinical pathologist (H. Haga).
Immunoprecipitation–Western Blot Analysis Mouse Abl1b cDNA tagged with hemagglutinin (HA) sequence at
the C-terminus (ABL1b-HA) was mutagenized by the QuikChange
Lightning Multi Site-Directed Mutagenesis Kit (Agilent) to derive KD
ABL1b(K290R)-HA mutant. These ABL1b constructs were placed into
the pEFBosneo expression vector ( 52 ). DAB1 -FLAG cDNA was inserted
into pcDNA3 (Invitrogen). TRIO cDNA ( 53 ) was tagged with T7 sequence
at the N-terminus and inserted into expression vector pCX ( 54 ). These
constructs were transfected into colorectal cancer or HEK293T cells, and
immunoprecipitation–Western blot analysis was performed as described
( 3 ) using agarose conjugated with anti-HA, anti-FLAG, or anti-T7 anti-
bodies (MBL), and antibodies for HA, FLAG, phosphotyrosine (pY; Cell
Signaling Technology), TRIO pY1990, TRIO pY2681, or T7 (Novagen).
Endogenous DAB1, ABL, and TRIO were immunoprecipitated
with anti-DAB1, anti-ABL, and anti-TRIO antibodies (Santa Cruz
Biotechnology), respectively, from lysates of the mouse intestines.
Immunocytochemistry HEK293T cells were transfected with expression plasmids for
T7-TRIO, ABL1b-HA, and DAB1-FLAG, stained for T7 and Tyr-phos-
phorylated TRIO pY1990 or TRIO pY2681, and mounted under cov-
erslips with VECTASHIELD Mounting Medium with DAPI (Vector
Laboratories).
Active RHO GTPases Pull-Down Assays RHO-GTP or RAC-GTP was detected using a RHO activation assay
kit (Thermo) or a RAC1 activation assay kit (Millipore), respectively.
Colon26 TetON-AES-FLAG mouse cancer cells ( 3 ) in which AES was
induced for 24 hours by doxycycline were cultured in monolayers,
scratched in multiple parallel lines, incubated further for 16 hours,
and subjected to the RHO/RAC pull-down assays.
TRIO Mutagenesis Possible Tyr phosphorylation sites in human TRIO were predicted
by NetPhos2.0 online software ( 22 ). To create unphosphorylatable
YF mutants, we mutagenized 18 Tyr residues in TRIO WT with high
pY probabilities (>0.5) to Phe.
Preparation of Specifi c Antibodies for TRIO pY1990and TRIO pY2681
Phospho-peptides VRDLG(pY)VVEG (residues 1985–1994) and
NPNYI(pY)DVPPE (residues 2676–2686) were synthesized and
injected into rabbits or chickens at SCRUM (Japan). Each antibody
preparation was affi nity-purifi ed using the immobilized antigen and
the corresponding unphosphorylated peptide. Titers of the affi nity-
purifi ed fractions were confi rmed by ELISA. Antibodies from either
species produced essentially the same results.
Construction of TetON Cells RKO TetON cells were established using pCMV-Tet3G and pTRE3G
(Clontech) containing cDNAs for ABL1b-HA, DAB1-FLAG, or T7-TRIO.
RHOGEF GTP Exchange Assay In Vitro We transfected HEK293T cells with pCX-T7-TRIO (WT or Y2681F)
and pulled down the TRIO protein from cell lysates using anti–
T7-conjugated agarose (MBL). Then, we mixed the TRIO fraction
with recombinant RHOA or RAC1 in the presence of mant-GTP
(Cytoskeleton) that emitted stronger fl uorescence when bound to
RHO small GTPases.
Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.
Authors’ Contributions Conception and design: M. Sonoshita, M.M. Taketo
Development of methodology: M. Sonoshita, Y. Itatani, T. Terashima,
M.M. Taketo
Acquisition of data (provided animals, acquired and managed
patients, provided facilities, etc.): M. Sonoshita, Y. Itatani, F. Kakizaki,
Y. Sakai, M.M. Taketo
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): M. Sonoshita, Y. Itatani,
Y. Katsuyama, M.M. Taketo
Writing, review, and/or revision of the manuscript: M. Sonoshita,
Y. Itatani, T. Terashima, Y. Katsuyama, M.M. Taketo
Administrative, technical, or material support (i.e., report-
ing or organizing data, constructing databases): M. Sonoshita,
K. Sakimura, Y. Katsuyama, M.M. Taketo
Study supervision: M. Sonoshita, M.M. Taketo
Acknowledgments The authors thank H. Kikuchi and M. Ishida for excellent technical
assistance. They also thank the members in M.M. Taketo’s laboratory
and K. Kawada for advice and discussions; T. Honjo for the NICD
construct; K. Sanada for DAB1 constructs; N. Osumi and H. Imai for
advice on Dab1- mutant mice; T. Kitamura for the pMX-IRES-EGFP vec-
tor; M. Okabe for the pCX vector; S. Nagata for the pEFBosneo vector;
A. Debant and S. Schmidt for TRIO constructs; A. Kikuchi for the Tiam1
plasmid; S. Robine for TgvCr e ERT2 mice; S. Sumiyoshi, M. Fujimoto,
and H. Haga for help in IHC evaluation of clinical specimens; M. Mat-
sumoto, M. Shirane, and K. Nakayama for advice on TRIO phosphoryla-
tion; and H. Miyoshi and T. Stappenbeck for comments on this article.
Grant Support This work was supported in part by grants-in-aid from the Minis-
try of Education, Culture, Sports, Science and Technology (MEXT) of
Japan (to M. Sonoshita and M.M. Taketo) and grants from The Kanaé
Foundation for the Promotion of Medical Science and The Sagawa
Foundation for Promotion of Cancer Research (to M. Sonoshita).
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received June 9, 2014; revised November 18, 2014; accepted
November 19, 2014; published OnlineFirst November 28, 2014.
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2015;5:198-211. Published OnlineFirst November 28, 2014.Cancer Discovery Masahiro Sonoshita, Yoshiro Itatani, Fumihiko Kakizaki, et al.
RHOGEF Protein TRIO−ABL−DAB1−Activation of NOTCH Promotion of Colorectal Cancer Invasion and Metastasis through
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