Shinshu M ed J, 63⑸ :281〜293, 2015

Nox4‑generated ROS Regulate TGF‑β1‑induced
Motility of Colon Cancer Cells through the Low Molecular
Weight Protein Tyrosine Phosphatase‑Rho Signaling Pathway
Ye ZHANG , Jun NAKAYAMA and Tohru KAMATA
1) Department of Molecular Biology and Biochemistry, Shinshu University Graduate School of Medicine
2) Department of Molecular Pathology, Shinshu University Graduate School of Medicine
3) Division of Tumor and Cellular Biochemistry, Department of Medical Sciences, University of Miyazaki

We investigated the role of NADPH oxidase (Nox)4 in colon cancer development.The expression of Nox4,
unlike Nox1,was speciﬁcally induced in colon carcinoma and was quite similar to that of transforming growth
factor‑β (TGF‑β)1. TGF‑β1 treatment of colon cancer cells upregulated the Nox4 expression and ROS
production through SMAD3.Knockdown of Nox4 suppressed TGF‑β1‑induced cell motility.Nox4 modulated
the Rho activity through redox regulation of the low molecular weight protein tyrosine phosphatase (LM W ‑PTP)‑p190RhoGAP pathway upon TGF‑β1 stimulation. These ﬁnding suggests that Nox4 contributes to
TGF‑β1‑dependent motility of colon carcinoma through the LMW ‑PTP‑p190RhoGAP‑Rho signaling pathway. Shinshu Med J 63 : 281―293, 2015
(Received for publication March 23, 2015;accepted in revised form June 16, 2015)
Key words:Nox4, reactive oxygen species, colon cancer, TGF‑β1, cell migration
Abbreviations: ROS, reactive oxygen species; Nox, NADPH oxidase; LM W ‑PTP, low molecular weight protein
tyrosine phosphatase;TGF, transforming growth factor;SMAD3, mothers against decapentaplegic homolog 3

Introduction

mediated ROS formation contributes to proliferation of melanoma cells , motility of breast cancer

The NADPH oxidase (Nox) family represent

cells , and migration of glioblastoma cells . Withmajor cellular sources of ROS for signaling pur-regard to the relationships of Nox isoforms withposes and consist of Nox1 5 and Duox1 and 2 .

colon cancer, Nox1 has been extensively studied.

Among them,H O ‑generating Nox4 is localized to

Although Nox1 was shown to be upregulated in

the membrane of intracellular compartments suchcolon cancer cells

as the endoplasmic reticulum, mitochondria, andthe functional relevance to colon carcinogenesis still

perinuclear regions and is expressed in a variety ofremains under debate

tissues . While it exerts mediating roles in many

is abundant in the intestine, Nox4 is not expressed

physiological processes including vascularizationin the normal colon but is expressed to some extent

and glucose metabolism,aberrant control of Nox4‑

in a subset of colon cancer cell lines including

derived ROS has been implicated in human diseases

CaCO2 and SW620

including carcinogenesis . For example, Nox4‑

scriptionally controlled constitutive enzyme,alteredand to mediate their growth ,.In contrast to Nox1 which. Given that Nox4 is a tran-

expression of Nox4 may have signiﬁcant implicaCorresponding author:Tohru Kamata
Division of Tumor and Cellular Biochemistry,
Department of Medical Sciences,University of Miyazaki,

tions in carcinogenesis. In this regard, a recent
ﬁnding is noteworthy for demonstrating that Nox4

5200 Kihara, Kiyotake, Miyazaki 889 ‑1692, Japanis upregulated in rat colon tumors induced by aE‑mail:tohru kamata＠med.miyazaki‑u.ac.jp

heterocyclic amine carcinogen .More recently,theNo. 5, 2015

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Zhang .Nakayama.Kamata

elevated expression of Nox4 was detected in associ-of primary colorectal adenoma and adenocar-

ation with relapse in stage II‑colon cancer .These

cinoma patients who underwent surgical resection

observations raise the possibility that Nox4 might

at Shinshu University Hospital, Matsumoto, Japan

participate in some steps of colon cancer progres-from 1999 ‑2004 after informed consent was obtain-

sion,but the detailed mechanism of Nox4 action has

ed. These patients included 31 cases of tubular

not been deﬁned.

adenomas and 39 cases of adenoma/carcinomas

In this study, we evaluated the protein level ofwhich had not received any therapeutic agents or

Nox4 in human colon neoplasms with various grade

irradiation before surgery. Tissue samples fromof diﬀerentiation and investigated the Nox4 signa-

colorectal adenomas or carcinomas and the cut end

ling pathway involved in the colon cancer develop-of the normal‑looking mucosa were examined inment. Our results for the ﬁrst time demonstrated

this study. Histological diagnosis of colorectal can-that Nox4 overexpression,distinct from Nox1,wascer was made according to the World Health Orga-

induced in adenocarcinomas, and that Nox4‑gener-

nization histologic classiﬁcation.

ated ROS mediated TGF‑β1‑induced motility of

Immunohistochemistrycolon cancer cells by controlling the LMW ‑PTP‑

Formalin‑ﬁxed tissue samples were embedded inparaﬃn, deparaﬃnized, and retrieved in Tris‑

Rho axis.
Materials and M ethods
Cell lines and materials

EDTA pH8.0 by microwave for 30 min as described
previously . The tissue sections were stained with
the indicated antibodies by using immunoglobulins

HCT‑116 and RKO cells were obtained fromconjugated with horseradish peroxidase (HRP)

American Type Culture Collection (M anassas, VA,‑diaminoben(Dako Japan, Tokyo, Japan) and 3, 3ʼUSA).TGF‑β1 was purchased from R & D Systems

zidine tetrahydroxychloride (BD Bioscience, San‑

(M inneapolis, M N, USA), N‑acetylcysteine (NAC)

Jose, CA, USA). Counterstaining was performed

from Calbiochem (LaJolla, CA, USA), BIAM fromwith hematoxylin.

Molecular Probes (Eugene, OR, USA), rabbit anti‑

Scoring

Nox1 antibodies from Abcam (Cambridge, MA,

Tissue specimens in which ＞5% of the cells wereUSA), rabbit anti‑Nox4 antibodies (#58851) frompositive for each primary antibody were deﬁned as

Novus (Littleton, CO, USA), rabbit anti‑TGF‑β1

positive, and others were deﬁned as negative,

antibodies from BioVision (Mountain View, CA,according to criteria previously described . In theUSA),mouse anti‑phospho tyrosine and rabbit anti‑

positive group, immunohistochemical results were

Rho antibodies from Millipore (Tempecula, CA,

further categorized into 3 groups: weak (＋1),USA), rabbit anti‑p190RhoGAP antibodies frommedium (＋2), and strong (＋3) based on staining

Cell Signaling (Lake Placid, NY, USA), and rabbit

intensity and summarized in the histograms.

anti‑HA antibodies from Sigma Aldrich (St. Louis,

siRNAs and RT‑PCR

MO, USA). M ouse monoclonal anti‑Nox1 anti-

pSilencer‑hygro Nox4 siRNA vector was con-bodies were provided by dia DeXus (South San

structed as described previously . SMAD3 siRNAs

Francisco, CA, USA) and rabbit anti‑Nox1 anti-‑CUGUGUGAGUUCGCCUUCATT ‑3ʼ
(sense: 5ʼ,bodies by Dr. D. Lambeth. Rabbit anti‑Nox4 anti-‑UGAAGGCGAACUCACACAGTT‑
anti‑sense: 5ʼbodies were provided by Drs. A. Shah and J. Gold-3ʼ
) were synthesized. RT‑PCR was carried out asstein. pcDNA3.0‑Nox1, adenovirus (Adv) HA‑

described , and PCR was performed under the con-

LMW ‑PTP and HA‑LMW ‑PTP

dition of 35 cycles of 94 ℃ for 30 sec, 55 ℃ for 30

and GST‑PAK‑

1 expression vectors were described previously .

sec,and 72 ℃ for 1 min according to the protocol by

Tissue specimens of colorectal cancer

Promega (Madison, WI, USA). Noxs and SMAD3

Surgical specimens were obtained from 70 cases
282

primers (sense
Shinshu M ed J Vol. 63

Nox4‑generated ROS mediate migration of colon cancer cells‑GGAGCAGGAATTGGGGTCAC‑3ʼand antisense5ʼ

analysis was performed with Studentʼ
s t test.

‑TTGCTGTCCCATCCGGTGAG‑3ʼ
5ʼ
forNox1;sense

Diﬀerences with P‑values of ＜0.05 were considered‑CTCAGCGGAATCAATCAGCTGTG‑3ʼ
5ʼ
andantisense

to be statistically signiﬁcant.

‑AGAGGAACACGACAATCAGCCTTAG‑3ʼ for5ʼ

Results and Discussion‑
Nox4;sense 5ʼ
‑GGTCAAGAGCCTGGTCAAGA ‑3ʼ
and anti‑sense5ʼ

Diﬀerential expression of Nox4 and Nox1 in

TTGAAGGCGAACTCACACAG ‑3ʼfor SMAD3)

normal mucosa and adenoma‑carcinoma sequence

were used.

of human colon

Transfection and Virus infection

To address the role of Nox4 in colorectal cancer,

Lipofectamine 2000 (Invitrogen, Carlsbad, CA,

we ﬁrst performed comparative immunohisto-USA), Lipofectamine RNAi Max (Invitrogen), or

chemical analyses of Nox4 and Nox1 expression in

DharmaFECT (Waltham, MA, USA)were used fornormal colorectal mucosa as well as adenoma‑car-transfection..Infection with adenovirus vectors was

cinoma sequence during tumorigenesis of color-performed as described previously .

ectum. To date, no histological study is available

Measurement of ROS productionthat provides detailed relationships between Nox4

Cell suspensions were incubated with 200μM

expression and the grade of human colon epithelial

luminol and 1 unit horseradish peroxidase for 10

neoplasms. In contrast to Nox1 which is abundant

min at 37 ℃ as described . Luminescence wasin the normal colon, Nox4 expression was notquantiﬁed by Lmax (Molecular Devices,San Diego,

observed in either normal mucosa or tubular

CA, USA).

adenomas (Fig. 1A, 1C). However, unexpectedly, a

Rho activity assay

high level of Nox4 was expressed in the cytoplasmic

Cell lysates were pulled down by using GST‑

region of well and moderately diﬀerentiated

PAK1‑coupled glutathione‑sepharose beads, andadenocarcinomas, indicating that induction of thethe amount of active GTP ・Rho retained on the

Nox4 expression takes place at the advanced stages

beads was determined byimmunoblotting using anti‑of colorectal cancer (Fig. 1A, 1C). Immunoblotting

Rho antibodies as described .

analyses also showed that the expression of Nox4Labeling with BIAM

proteins was elevated in colon tumors compared toCells were lysed in phosphate buﬀer (pH 7.0)

adjacent normal tissues (Fig. S1),which is in agree-containing 20 mM NEM , an alkylator of cysteine‑

ment with the previous observation . We also

SH residues, reduced by 20 mM DTT and labeled

conﬁrmed that the two independent antibodies‑one

with 20μM BIAM as described . Labeled proteinsfrom Novus (Fig. 1A) and the other from Dr. J.

were analyzed by immunoprecipitation with anti‑

Goldstein (Fig. S2B)‑yielded a similar staining

HA antibodies, followed by SDS‑PAGE and HRP‑

pattern in normal mucosa and well‑diﬀerentiatedstreptavidin staining.

adenocarcinomas, which reinforces our ﬁnding of

Migration Assayspeciﬁc Nox4 expression associated with colorectal

M igration assay was performed by using Boyden

carcinoma.

chambers (BD Bioscience) as described previous-

Meanwhile, the precise relationships between thely . Cells were plated to the chambers of 24‑wellexpression pattern of Nox1 proteins and the grade

plates in serum‑free DMEM, and TGF‑β1 (10 ng/

of diﬀerentiation have not been examined,althoughml)was used as a cue.M igrating cells were counted

analysis of Nox1 mRNA expression in colon car-after 24h.

cinomas by in situ hybridization was previously

Statistical analysis

reported . Nox1 proteins were weakly detected

Data were shown as mean ± S.D.of the results ofalong lower crypts of normal mucosa (Fig. S3A),

at least three independent experiments. Statisticaland its expression level increased in association

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Fig. 1 Expression of Nox4 is upregulated in human colon tumors and correlates with expression of TGF‑β1.
(A) The specimens from human normal colon tissues, tubular adenomas (mild, moderate, and severe atypia), andadenocarcinomas (well diﬀerentiated, moderately diﬀerentiated, and poorly diﬀerentiated type) were subjected toimmunostaining with rabbit anti‑Nox4 antibodies from Novus.(B)The specimens from normal colon tissues,tubular
adenomas, and adenocarcinomas (well diﬀerentiated and moderately diﬀerentiated type)were immunostained withanti‑TGF‑β1 antibodies. (C) Frequency of Nox4 expression in normal colon tissues and colon tumor samples.
Samples from 10 patients were examined for each grade of tumor and normal tissues.Frequency of Nox4 expression
was scored as described in Materials and M ethods. The histograms represent mean ± S. D. (n＝10). (D)Frequencyof the TGF‑β1 expression in colon tumors was estimated as in the case of Nox4 (see M aterials and M ethods).The
histograms represent mean ± S.D. (n＝5).

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Nox4‑generated ROS mediate migration of colon cancer cellswith the degree of atypia of tubular adenomas (Fig.

veriﬁed by immunoblotting analysis of the protein

S3A, S3B). The expression of Nox1 proteins rea-levels of Nox4 and Nox1 (Fig. S4). Furthermore,ched a maximum level in the apical cell surface ofpretreatment of RKO cells with SIS3, a speciﬁcwell diﬀerentiated adenocarcinomas but decreased

inhibitor of the SMAD3 transcription factor

as cancer progressed through moderately and poor-

signiﬁcantly suppressed the TGF ‑β1 ‑induced

ly diﬀerentiated adenocarcinomas (Fig. S3A, S3B).

expression of Nox4 mRNAs (Fig. 2B).Knockdown

Taken together, the data revealed the strikingof SMAD3 by siRNAs also suppressed the expres-diﬀerence in expression pattern between Nox4 andsion of both Nox4 mRNAs and Nox4 proteins (Fig.

Nox1 during colon cancer development :Nox4 wasS5). Thus, we conclude that TGF‑β1 regulates the

speciﬁcally expressed at the stages of colon car-Nox4 expression through the SMAD3 pathway incinomas,while increased expression of Nox1 occur-colon cancer cells.

red even in adenomas during neoplastic progression.

In addition, we found that TGF‑β1 treatment

TGF‑β1 is expressed in colon cancer andenhanced ROS generation in the cells, and that theinduces both Nox4 expression and ROS production

stimulatory eﬀect of TGF‑β1 on ROS synthesis was

We next examined the regulatory mechanism of

suppressed by transduction of SMAD3 siRNAs or

Nox4 induction, which is closely associated withaddition of SIS3 (Fig. 2C). RT‑PCT and immunob-

progression of colon carcinomas. Gene expressions

lotting analyses indicated that SMAD3 siRNAs

are frequently inﬂuenced by stimuli such as cyto-

suppressed the expression of SM AD3(Fig.2D).The

kines and growth factors, which are secreted fromresults support that the view that TGF‑β1 stimu-

the colon cancer cells and surrounding stromal cells,

lates ROS production by inducing Nox4 expression

via either autocrine or paracrine signalings.Among

via SMAD3.

them, TGF‑β1 acts as a stimulator of both cell
growth and invasion during colorectal cancer pro-

Nox4 mediates TGF‑β1‑induced migration ofcolon cancer cells

motion, and its intense expression correlates with

Because Nox4 but not Nox1 was found to partici-

pathological progression to metastasis and poor

pate in TGF‑β1 signaling, we focused on the

prognosis . We therefore reasoned that TGF‑β1

regulatory role of Nox4 signaling in TGF‑β1‑

might be involved in the regulation of Nox4 expres-dependent cell motility in the subsequent study.We

sion in colon carcinoma.To test this possibility,weﬁrst examined the involvement of Nox4 in TGF‑β1‑

evaluated the expression pattern of TGF‑β1 ininduced migration of colorectal cancer cells. Bothnormal colon and tumor samples.Interestingly,well

HCT‑116 and RKO cells were treated with TGF‑β1

and moderately diﬀerentiated adenocarcinomas ex-

following transfection of Nox4‑speciﬁc siRNAs or

hibited strong staining of TGF‑β1,whereas normal

scrambled siRNAs and tested for their ability tomucosa and tubular adenomas displayed no TGF‑

migrate. The number of migrating cells was markβ1 staining (Fig. 1B). Of note, the staining patternedlyenhanced byTGF‑β1 treatment,whereas TGF‑

of TGF‑β1 remarkably resembled that of Nox4 in

β1‑promoted cell migration was attenuated byterms of association with grade of colon carcinoma

Nox4 knockdown (Fig. 3A).Immunoblotting analy-

and the localization (Fig.1A‑1D).This prompted us

sis demonstrated eﬃcient ablation of endogenous

to determine whether TGF‑β1 induces Nox4

Nox4 by Nox4 siRNAs (Fig. 3B).The data point toexpression. To this end, RKO and HCT‑116 colon

an important role of Nox4‑dependent mechanism incarcinoma cells were treated with TGF‑β1 andTGF‑β1‑induced motility of colon cancer cells.

subjected to analysis of Nox4 expression by RT‑

To further analyze the Nox4 signaling pathway

PCR. TGF‑β1 speciﬁcally upregulated the expres-leading to cell motogenesis, we ﬁrst determinedsion of Nox4 mRNAs, whereas the expression ofwhether Nox4‑generated ROS modulate the activ-

Nox1 was not aﬀected (Fig. 2A). This was also

ityof Rho,a keyregulator of cytoskeletal contracti-

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Fig. 2 TGF‑β1 induces both Nox4 expression and Nox4‑derived ROS production in colon cancer cells.
(A)RKO and HCT 116 cells were treated with TGF‑β1 (10 ng/ml)for 24h.Expressions of Nox1 and Nox4
mRNAs were analyzed by RT‑PCR.(B)RKO cells were treated with TGF‑β1 (10 ng/ml)in the absence orpresence of a SMAD3 inhibitor, SIS3 (10μM ) for 24 h and subjected to RT‑PCR analysis. (C) RKO andHCT116 cells (5x10 )were treated with TGF‑β1 and SIS3 as in (B)or transfected with SM AD3 siRNAs andscrambled siRNAs(sc)prior to TGF‑β1 treatment for 24h. The cells were then subjected to luminol‑based
ROS assay.The data represent mean ± S.D (n＝3)in 3 separate experiments.P1 or P2 ＜0.05 versus sc,TGF‑
β1. (D)RKO and HCT116 cells were transfected with SM AD3 siRNAs or scrambled controls, treated with
TGF‑β1 as described in (C), and subjected to RT‑PCR and immunoblotting with rabbit anti‑SM AD3
antibodies. EF1α and β‑actin are loading controls.

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Nox4‑generated ROS mediate migration of colon cancer cells

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Fig. 3 Nox4 mediates TGF‑β1‑induced migration of colon cancer cells.
(A)RKO and HCT‑116 cells were transfected with Nox4 siRNAs or scrambled siRNAs. Transfected cells
were replated 48h later, treated with TGF‑β1 (10 ng/ml), and subjected to migration assay as described in
M aterials and Methods.The numbers of migrating cells were determined.The data represent mean ± S.D.
(n＝3) in three separate experiments. (B) Alternatively, the expression of Nox4 in transfected cells wasanalyzed by immunoblotting with anti‑Nox4 antibodies. β‑actin is a loading control.

lity. GST‑Rho binding domain pull‑down assays

β1 treatment elevated tyrosine phosphorylation ofindicated that the amount of active GTP‑bound

p190RhoGAP, whereas Nox4 siRNAs abolished theRho was decreased upon TGF‑β1 treatment ofstimulatory eﬀect of TGF‑β1 on p190RhoGAP

RKO cells (Fig. 4A),whereas the suppressive eﬀect

phosphorylation.This suggests that Nox4 mediatesof TGF‑β1 on Rho was abolished when cells were

TGF‑β1‑induced activation of p190RhoGAP (Fig.

transfected with Nox4 siRNAs (Fig. 4A). Consis-

4B), although the nature of a putative tyrosine

tently, SIS3 and NAC, an antioxidant, interferedkinase involved is unknown at present.

with the suppressive action of TGF‑β1 on Rho (Fig.

Given that low molecular weight‑protein tyrosine‑S6). These results indicate that Nox4 signaling

phosphatase (LM W ‑PTP) deactivates tyrosine‑

mediates TGF‑β‑induced cell motility by negative-

phosphorylated p190RhoGAP

ly regulating the Rho activity.

vates LMW ‑PTP through oxidation of its redox‑

and H O inacti-

p190RhoGAP is activated upon tyrosine phosphor-sensitive Cys‑12 and ‑17 residues in its catalytic

ylation by receptor or non‑receptor tyrosine

pocket , we next investigated whether Nox4‑gen-kinases, thereby down‑regulating Rho . We there-erated ROS transmit an activation signal tofore speculated that TGF‑β1‑mediated negative

p190RhoGAP via LMW ‑PTP. To this end,we test-

regulation of Rho could be caused by activation ofed the ability of Nox4 to oxidize LM W ‑PTP.Cells

p190RhoGAP in response to TGF‑β1 stimulation.

were transfected with LM W ‑PTP, and cell lysates

Immunoblotting analyses demonstrated that TGF‑

were processed for BIAM labeling,in which redox‑

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1

1

S iNox4

S iNox4

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1

Fig.4 Nox4 mediates TGF‑β1‑induced cell migration via the LM W ‑PTP‑p190RhoGAP‑Rho pathway.
(A)TGF‑β1 downregulates the Rho activity through Nox4.RKO cells were transfected with scrambled siRNAs
or Nox4 siRNAs,treated with TGF‑β1 (10 ng/ml)for 10 min,and processed for cell lysate preparation.Lysates
were subjected to the Rho activity assay as described in M aterials and M ethods.The total Rho was monitored
by immunoblotting with anti‑Rho antibodies. (B)RKO cells were transfected with scrambled siRNAs or Nox4
siRNAs and treated with TGF‑β1(10 ng/ml)for 10 min.Lysates were subjected to immunoblotting analysis withanti‑phospho‑p190RhoGAP and p190RhoGAP antibodies.(C)RKO cells were transfected with pcDNA3.0‑Nox4
or control vectors, infected with adv‑HA‑LMW ‑PTP, and 48h later treated with or without NAC (10mM)for
30 min. BIAM labeling of LMW ‑PTP, following alkylation and reduction, was performed as described in
M aterials and Methods. Expressions of transfected LMW ‑PTP and Nox4 were monitored by immunoblottingwith anti‑HA antibodies and anti‑Nox4 antibodies. (D)RKO cells were transfected with scrambled siRNAs or
Nox4 siRNAs,infected with adv‑HA‑LMW ‑PTP
or control viruses,and subjected to migration assay using
TGF‑β1 (10 ng/ml). Expression of introduced LMW ‑PTP
was monitored by immunoblotting with anti‑HA
antibodies.The data represent mean ± S.D.(n＝3)in four separate experiments.(E)Schematic model for Nox4‑
mediated TGF‑β1‑induced cell migration. TGF‑β1 induces Nox4 expression possibly in a SM AD3‑dependent
manner. Nox4‑derived ROS oxidize and inactivate LMW ‑PTP, which results in accumulation of activated,tyrosine phosphorylated p190RhoGAP. This causes downregulation of Rho, leading to cell migration of coloncancer cells.

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Nox4‑generated ROS mediate migration of colon cancer cells

sensitive cysteine residues oxidized by intracellularmore, Nox4 mediated TGF‑β1‑triggered motility

H O are protected from postlysis alkylation withof colon cancer cells via the LMW ‑PTP‑Rho path-

NEM and reacted with BIAM following reduction

way.Supporting this observation,Nox4 is known to. Co‑transfection of Nox4 promoted

localize to focal adhesions, thereby coordinating

BIAM labeling of LMW ‑PTP, and treatment withcell adhesion and migration together with Src

NAC decreased the labeling (Fig. 4C). Introduction

tyrosine kinases . We postulate that TGF‑β1

of Nox4 siRNAs also attenuated BIAM ‑labeling of

induces the Nox4 expression in the colon cancer

LMW ‑PTP in RKO cells (Fig. S7). This is consis-

cells, and that Nox4‑derived ROS subsequently

tent with the notion that LMW ‑PTP is oxidized bytransmit TGF‑β1 signaling through theLMW ‑PTP‑

Nox4‑generated ROS, serving as their sensor. To

RhoGAP‑Rho axis to accelerate cell migration,

test the involvement of LM W ‑PTP in TGF‑β1‑

thereby contributing to the invasiveness of colon

induced cell migration, cells were transfected withcancer cells (Fig. 4E).Similar to our ﬁnding,TGF‑

Nox4 siRNAs and/or LM W ‑PTP

β1‑secreting stromal cells activated Nox4‑depen-with DTT,a catalytical-

ly inactive, Cys‑12 to Ser mutant of LM W ‑PTP

dent migration of breast cancer cells when cocultur-and subjected to cell migration assay. Stimulation

ed .Although Bauer KM et alof migration with TGF‑β1 was blocked in Nox4

involvement of Nox4 in the motilityof colon cancer,

siRNA‑transfected cells,whereas forced expression

no detailed mechanistic study has been provided.

of LM W ‑PTP

restored TGF ‑β1‑induced

Thus, our study is signiﬁcant in the sense that it

motogenesis in Nox4 siRNA‑transfected cells (Fig.

revealed the Nox4 signaling mechanism responsible

4D).The motogenic eﬀect of TGF‑β1 on scrambled

for the motility of colorectal cancer.

siRNA‑transfected cells was not signiﬁcantly altered by introduction of LM W ‑PTP

Conﬂict of Interest, possibly

because endogenous LMW ‑PTP had been inactivated byTGF‑β1 via Nox4.The data demonstrate that
LMW ‑PTP mediates TGF‑β1‑induced, Nox4‑
dependent cell migration.

have suggested the

The authors declare no conﬂict of interest in thiswork.
Acknowledgments

In summary, our study revealed that Nox4 is

We thank Drs. S. Miyagawa and R. Hiraga fordiﬀerentially expressed from Nox1 during theproviding tissue samples. We also thank Drs. A.

course of colon tumor development :Nox4 expres-Shah and J. Goldstein for providing the Nox4 anti-sion is speciﬁcally induced in colon carcinomas,

bodies, Dr. D. Lambeth for providing the Nox1

while Nox1 expression begins to increase in

antibody, and Dr. D. Koinuma for providing the

adenomas even before malignant colon tumor.

SMAD3 antibody. We are grateful to F. Ushiyama

Another important ﬁnding is that Nox4 expression

for assistance in manuscript preparation.This workin colon primary tumors is upregulated similar towas supported by a Grant on Cancer Research inthat of TGF‑β1, and that Nox4 but not Nox1 isApplied Areas from the Ministry of Science andinduced by TGF‑β1 in colon cancer cells. Further-

Culture of Japan (T. K.:18012019).

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Nox4‑generated ROS mediate migration of colon cancer cells
Fig. S1 Immunoblotting analysis of Nox4 expression in colon tumors.
Fractionation of colon tissues and immunoblotting analysis using anti‑Nox4 antibodies were performed as described
previously［1］. Cell lysates were prepared from normal colon tissues (N) or colon tumors (well diﬀerentiatedadenocarcinomas)(T)from three patients. β‑actin is a loading control. ＊indicates the degraded Nox4 proteins.
［1］ Komatsu D, Kato M , Nakayama J, Miyagawa S, Kamata T :NADPH oxidase 1 plays a critical mediating rolein oncogenic Ras‑induced vascular endothelial growth factor expression. Oncogene 27:4724‑4732, 2008
Fig.S2 Validation of immunohistochemical data on Nox4 and Nox1 expressions in colon tumors by using alternative
sources of the antibodies.
To verify the immunohistochemical data on Nox4 (Fig. 1A) and Nox1 (Fig. S2A) expressions, tissue sections fromnormal colon and well‑diﬀerentiated adenocarcinomas were immunostained with the rabbit anti‑Nox4 antibody
provided by Dr. J. Godstein, Thomas Jeﬀerson University (A) and the rabbit anti‑Nox1 antibody provided by Dr. J.
Lambeth, Emory University (B). The data indicate that Dr. Goldsteinʼs antibodies and Dr. Lambethʼs antibodies
exhibited staining patterns similar to those obtained with the Novus Nox4 antibody(Fig.1A)and the dia DeXus Nox1
antibody (Fig. S2), respectively.
Fig. S3 Expression of Nox1 in human colon tumors.
(A) The specimens from human normal colon tissues, tubular adenomas (mild, moderate, and severe atypia), andadenocarcinomas (well diﬀerentiated, moderately diﬀerentiated, and poorly diﬀerentiated type) were subjected toimmunostaining with mouse monoclonal anti‑Nox1 antibodies.(B)Frequency of the Nox1 expression in normal colon
tissues and colon tumor samples. Samples of 10 patients were examined for each grade of tumors and normal tissues.
Frequency of the Nox1 expression was scored as described in M aterials and Methods. The histograms represent
mean ± S. D. (n＝10).
Fig. S4 TGF‑βinduces the expression of Nox4 proteins.
RKO and HCT116 cells were treated with TGF‑β1 (10 ng/ml)for 24h, and expressions of Nox4 and Nox1 proteins
were analyzed by immunoblotting with rabbit anti‑Nox4 antibody provided by Dr. A. Shah and rabbit anti‑Nox1
antibody from Abcam, respectively. The data indicated that Nox4 proteins but not Nox1 proteins were induced by
TGF‑β1, which is in agreement with the results of RT‑PCR analysis (Fig. 2). β‑actin is a loading control.
Fig. S5 Knockdown of SMAD3 suppresses TGF‑β1‑induced expression of Nox4 mRNAs and Nox4 proteins.
RKO cells were transfected with SMAD3 siRNAs or scrambled siRNAs (se), 48h later treated with TGF‑β1 (10 ng/ml) for 24h, and subjected to both PCR analysis and immunoblotting using rabbit‑anti Nox4 antibodies from Dr.
Goldstein.The data indicated that knockdown of SMAD3 by siRNAs suppressed TGF‑β1‑induced expression of Nox4
mRNAs andNox4 proteins, which is consistent with the results of RT‑PCR analysis (Fig. 2B).
Fig. S6 SIS3 and NAC block TGF‑β1‑dependent regulation of Rho.
RKO cells were treated with TGF‑β1 (10 ng/ml)in the presence or absence of SIS3 (10μM)for 24h or NAC (10mM)
for 30 min and subjected to the Rho activity assay as described in M aterials and Methods.The total Rho was monitored
by immunoblotting.The data indicated that treatment with SIS3 and NAC abolished TGF‑β1 ‑induced suppression of
the Rho activity,which is consistent with the idea that TGF‑β/SMAD3‑induced Nox4 mediates the regulation of Rho.
Fig. S7 Nox4 knockdown suppresses oxidation of LM W ‑PTP.
RKO cells were transfected with pSilencer‑Nox4 siRNA or pSilencer‑scrambled siRNA vectors and infected with
Adv‑HA‑LMW ‑PTP.48h later,cell lysates were prepared and subjected to BIAM labeling of LM W ‑PTP as describedin M aterials and Methods.Expressions of transfected LM W ‑PTP and Nox4 were monitored as in Fig.4C and Fig.S4,
respectively.

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