Promotion of Colorectal Cancer Invasion and Metastasis ...when colorectal cancer cells with...

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

Research. on January 11, 2021. © 2015 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst November 28, 2014; DOI: 10.1158/2159-8290.CD-14-0595

http://cancerdiscovery.aacrjournals.org/

FEBRUARY 2015�CANCER DISCOVERY | 199

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




200 | CANCER DISCOVERY�FEBRUARY 2015 www.aacrjournals.org

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)

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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)

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0 (kb)

0

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H. sapiens

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RBPJ-binding

sequences

High affinity (YGTGRGAA)

Low affinity (RTGRGAR)

Primer pair for ChIP q-PCR

mu

Pro

port

ion (

%)

#

#

*

*

*

* **

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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





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)

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100

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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

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DAB1 – DAB1 – DAB1

Rela

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l tu

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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






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

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ABL1b-HA

HAIP: HA

IP: lgG lgG

anti-

DAB1

anti-

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DAB1-FLAG

IP: FLAGFLAG

0

WT WT WT

WT

– – –

Imatinib [ABL inh.] (μmol/L)0.1 101

*

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**

**

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**

Num

ber

of cells

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ded

(per

×400 fie

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of cells

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×200 fie

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of cells

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(per

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siA

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siA

BL2

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WB:

ABL

DAB1

1 2 3 4 5 6 7 8 9 10 11

pY

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KD

Apc –/–Aes –/– (Apc/Aes) tumors

– – –






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

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0.5 ≤ φ < 2 2 ≤ φ

Invasion depth ofApc/Aes tumors

Apc/Aes tumor size

Imatinib

ImatinibVehicleImatinib

Tumor diameter (mm)Shallow → Deep






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






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






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

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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






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






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 ).






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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