Hepatocyte Growth Factor Activator Inhibitor Type 1 (Hai-1/Spint1) Is a Suppressor of Intestinal Tumorigenesis

Author Manuscript Published OnlineFirst on February 27, 2013; DOI: 10.1158/0008-5472.CAN-12-3337 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Hepatocyte growth factor activator inhibitor type 1 (Hai-1/Spint1) is a suppressor of intestinal tumorigenesis

Shinri Hoshiko,*,# Makiko Kawaguchi,* Tsuyoshi Fukushima,* Yukihiro Haruyama,*

Kenji Yorita,* Hiroyuki Tanaka,* Motoharu Seiki,‡ Haruhiko Inatsu,# Kazuo Kitamura#

and Hiroaki Kataoka*

*Section of Oncopathology and Regenerative Biology, Department of Pathology and

#Section of Circulatory and Body Fluid Regulation, Department of Internal Medicine,

Faculty of Medicine, University of Miyazaki, Miyazaki, Japan; ‡Division of Cancer Cell

Research, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

S.H. and M.K. contributed equally to this study

Running title: HAI-1 suppresses intestinal tumorigenesis

Key words: HAI-1, carcinogenesis, colon cancer, hepatocyte growth factor, epithelial

integrity

Financial support: This work was supported by Grant-in-Aid for Scientific Research

no. 24390099 (H.K.) and no. 23790250 (M.K.) from the Ministry of Education, Science,

Sports and Culture, Japan.

Corresponding author: Hiroaki Kataoka, Section of Oncopathology and Regenerative

Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, 5200

Kihara, Kiyotake, Miyazaki 889-1692, Japan. Phone, +81-985-852809; Fax,

+81-985-856003; E-mail, [email protected]

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 27, 2013; DOI: 10.1158/0008-5472.CAN-12-3337 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Disclosure Statement: The authors have no conflict of interest.

The whole raw data of microarray analysis for gene expression are available for review

at our web site: http://www.med.miyazaki-u.ac.jp/patho2/englishpage/official2.html and

will be available at GEO when the manuscript is accepted for publication.

Word count (text): 4480

Total number of figures and tables: 6 (5 figures and 1 table)

Abstract

Hepatocyte growth factor activator inhibitor type 1 (HAI-1/SPINT1) is a

membrane-bound serine protease inhibitor expressed on the surface of epithelial cells.

Although HAI-1/SPINT1 is abundantly expressed in the intestinal epithelium, its role in

intestinal tumorigenesis is not known. In this study, we investigated the role of

Hai-1/Spint1 in intestinal tumorigenesis using mouse models. The membranous

Hai-1/Spint1 immunoreactivity was decreased in murine ApcMin/+ tumors and also in

carcinogen (azoxymethane treatment followed by dextran sodium sulfate

administration)-induced colon tumors compared to the adjacent non-neoplastic

epithelium. The decreased immunoreactivity appeared to be due to sheddase activity

of membrane-type 1 matrix metalloprotease. Then, we examined the effect of

intestine-specific deletion of Spint1 gene on ApcMin/+ mice. The loss of Hai-1/Spint1

significantly accelerated tumor formation in ApcMin/+ mice and shortened their survival

periods. Activation of hepatocyte growth factor was enhanced in

Hai-1/Spint1-deficient ApcMin/+ intestine. Gene expression profiling revealed

upregulation of the Wnt/-catenin-signaling circuit, claudin-2 expression, and

angiogenesis not only in tumor tissue but also in the background mucosa without

macroscopic tumors in Hai-1/Spint1-deficient ApcMin/+ intestine. Intestinal deletion of

Spint1 also enhanced the susceptibility to carcinogen-induced colon tumorigenicity of

wild-type Apc mice. Our findings suggest that HAI-1/SPINT1 has a crucial role in

suppressing intestinal tumorigenesis, which implies a novel link between epithelial cell

surface serine protease inhibitors and protection from carcinogenic stimuli.

(216 words)

Introduction The intestinal mucosal surface area represents the most significant anatomic and

physiological portal for systemic exposure to environmental toxins and microorganisms

(1). The systemic compartment is protected from these exposures by the mucosal

epithelial barrier, with barrier dysfunction contributing to local and systemic diseases,

including inflammatory bowel disease, allergic disorders and malignancies (1, 2).

Regarding the epithelial barrier, the role for the junctional complexes that restrict

paracellular macromolecular exchange between mucosal and systemic compartments

has been studied extensively and the molecular components of tight junction proteins

described (1, 2). However, how the cell surface proteolysis systems participate in

maintaining epithelial barrier function remains incompletely defined. Recent evidence

indicates that normal functioning of the cell surface serine protease matriptase is

critically required for epithelial integrity (3-6). Matriptase, encoded by the ST14 gene,

belongs to the type II transmembrane serine protease (TTSP) superfamily and is

expressed on the epithelial cell surface of most epithelial tissues (6). Moreover, recent

studies indicate that its cognate cell surface inhibitor, hepatocyte growth factor activator

inhibitor (HAI), also has crucial roles in epithelial functions (7-10).

HAI belongs to the type I family of transmembrane serine protease inhibitors that

consists of two members: HAI type 1 (HAI-1)/serine protease inhibitor Kunitz type 1

(SPINT1) and HAI-2/SPINT2, which both have two extracellular Kunitz domains and a

transmembrane domain and are encoded by the SPINT1 and SPINT2 genes, respectively

(11). Unlike HAI-2/SPINT2, HAI-1/SPINT1 also has an N-terminal cysteine-rich

domain and a low density lipoprotein receptor type A-like domain (11).

HAI-1/SPINT1 is expressed in most epithelial cells, with significant expression in

enterocytes (8, 12). HAI-1/SPINT1 was initially discovered as an endogenous

inhibitor of hepatocyte growth factor (HGF) activator, a serum-derived serine protease

(13), but its membrane association may also allow it to regulate cell surface serine

proteases. Indeed, ample evidence indicates that HAI-1/SPINT1 is a cognate inhibitor

of matriptase on epithelial cell surfaces (10, 14-17). Other TTSPs, such as hepsin,

human airway trypsin-like protease, TMPRSS4 and TMPRSS13, as well as the

glycosylphosphatidylinositol-anchored cell surface serine protease prostasin, may also

be regulated by HAI-1/SPINT1 (6, 18-20). Therefore, HAI-1/SPINT1 must have a

critical regulatory role in pericellular proteolysis of epithelial cells. In fact,

HAI-1/SPINT1 has essential functions in placental trophoblasts, epidermal

keratinocytes, and hair follicle epithelium (7, 21). Recently, we reported that

conditional deletion of the Spint1 gene in intestinal epithelial cells results in enhanced

intestinal permeability and susceptibility to dextran sulfate sodium (DSS)-induced

colitis (8). Since intestinal epithelial integrity has an important protective role against

mucosal inflammation and neoplastic progression (1, 2, 22), HAI-1/SPINT1 may have a

significant role in intestinal tumorigenesis. Moreover, all major physiological HGF

activators (matriptase, hepsin, HGF activator) are sensitive to HAI-1/SPINT1 and

previous study revealed that colorectal cancer cells show decreased cell surface

HAI-1/SPINT1 immunoreactivity with enhanced HGF activation in the cancer tissue

(23). However, little is known about how HAI-1/SPINT1 may participate in intestinal

tumorigenesis.

In this study, we evaluated the role of HAI-1/SPINT1 in intestinal tumorigenesis

using intestinal specific Hai-1/Spint1 knockout mice (8) in two intestinal carcinogenesis

mouse models: an Apc mutant mouse and two-step chemical carcinogenesis in mouse

colon. Our results from both models confirm that Hai-1/Spint1 has a critical role in

suppressing intestinal tumorigenesis.

MATERIALS AND METHODS

Animal Experiments

All animal studies were reviewed and approved by the Committee on the Ethics of

Animal Experiments of the University of Miyazaki Animal Research Committee, in

accordance with international guidelines for biomedical research involving animals.

All mice were housed in a pathogen-free barrier environment for the duration of the

study. ApcMin/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME).

Mice with intestinal epithelial cell-specific deletion of the Spint1 gene

(Spint1flox/flox/Vil-Cre) were generated by interbreeding mice carrying Spint1/LoxP

homozygous alleles (Spint1flox/flox) with Cre transgenic mice under the control of the

intestine-specific villin promoter (8). ApcMin/+ mice were then interbred with

Spint1flox/flox/Vil-Cre mice, generating ApcMin/+ mice with intestine-specific deletion of

the Spint1 gene (ApcMin/+/Spint1flox/flox/Vil-Cre). All mice had a C57BL/6 genetic

background and were fed a semi-purified AIN-76 diet under specific pathogen-free

condition. For ApcMin/+ tumorigenesis assays, age and sex-matched mice were selected

to assess tumor formation in the small and large intestine. At 15 weeks of age, all

mice (male, n=19 and 15 for ApcMin/+/Spint1flox/flox/Vil-Cre and ApcMin/+/Spint1flox/flox,

respectively) were sacrificed by diethyl ether inhalation and the numbers and major

diameters of intestinal polyps were determined. The polypoid tumor lesion size was

scored as follows: <1 mm, score 1; <2 mm, score 2; <3 mm, score 3; >3 mm, score 4.

For the chemical carcinogenesis study, Spint1flox/flox/Vil-Cre mice and Spint1flox/flox

control mice were used for combined treatment with azoxymethane (AOM) and DSS

(24). At 8 weeks of age all mice (male, n=18 and 23 for Spint1flox/flox/Vil-Cre and

Spint1flox/flox, respectively) were treated with a single intraperitoneal injection of AOM

(Sigma-Aldrich, St. Louis, MO) (10 mg/kg body weight). One week after the AOM

injection, mice were exposed to 1% DSS (molecular weight 36-50 kDa; MP

Biomedicals, Solon, OH) in the drinking water for 5 days and thereafter received no

further treatment. All mice were sacrificed 20 weeks after the AOM treatment. For

histological analysis, formalin-fixed paraffin embedded tissue sections were stained

with H&E or processed for immunohistochemistry.

Immunohistochemistry, immunofluorescence, immunoblotting and gene expression

analysis

The following antibodies were used: anti-human HAI-1 monoclonal antibody 1N7

(12), anti-mouse Hai-1 goat polyclonal antibody raised against recombinant Hai-1

lacking transmembrane and intracytoplasmic domains (R&D Systems, Minneapolis,

MN), anti-mouse/human Hai-1 rabbit polyclonal antibody raised against

intracytoplasmic domain (Pro484-Leu507) (25), anti-mouse/human matriptase

(AnaSpec, San Jose, CA), anti-mouse Hgf (R&D Systems), anti-mouse -catenin

(Sigma-Aldrich), anti-phosphorylated c-MET (Y1235) (26), and anti-mouse CD31

(AnaSpec) rabbit polyclonal antibodies. The protocol for immunohistochemistry of

paraffin-embedded sections of surgically resected colorectal tumors (4 cases) was

approved by the ethical board of the Faculty of Medicine, University of Miyazaki.

The immunohistochemistry methods used were described previously (12, 26, 27).

Immunofluorescence and immunoblot analyses were performed as described (8).

Preparation of Hgf-enriched tissue extract and analysis of Hgf processing by

immunoblot were performed as described previously (23). Reverse transcription

(RT)-PCR and quantitative real-time RT-PCR (qRT-PCR) were performed using the

primer sequences and methods described in Supplementary Table 1. Microarray

analysis was performed as described previously (27) using a GeneChipTM Mouse

Genome 430 2.0 Array (Affymetrix, Santa Clara, CA).

Cell culture, gene knockdown and forced expression

The MT1rev2 cell line was established from Mmp14 null mice intestine as

described previously (28). MT1rev2 cells carry an exogenous mouse Mmp14 gene

whose expression is suppressed in the presence of doxycycline (1 g/mL; Sigma) so

that cells express MT1-MMP/Mmp14 in doxycycline-free medium. To detect secreted

Hai-1/Spint1, cells were washed three times with PBS and cultured in serum-free

medium without doxycycline for the indicated time period. The culture supernatant

was then concentrated in an Amicon-Ultra-4 (Millipore, Billerica, MA) and used for

immunoblotting

Statistics

Statistical analysis was done using GraphPad Prism, version 5.04 (GraphPad

Software Inc., San Diego, CA).

RESULTS

Decreased membranous HAI-1/SPINT1 immunoreactivity accompanies neoplastic

progression of intestinal epithelium

In accordance with previous observations (23, 25) the membranous

immunoreactivity of HAI-1/SPINT1 was markedly reduced in colon adenocarcinoma

cells compared to adjacent adenoma cells and non-neoplastic epithelium (Figure 1A).

This trend was also observed in a murine model of intestinal tumorigenesis mediated by

mutation of the Apc gene (ApcMin/+ mice) (Figure 1B). The neoplastic epithelium also

showed significantly decreased membranous Hai-1/Spint1 immunoreactivity compared

to the adjacent non-neoplastic epithelium. Notably, similar to human colon cancer

tissue (25), the Spint1 mRNA level was essentially preserved in ApcMin/+ tumor tissue

(Figure 1B), suggesting that the decreased immunoreactivity may be caused by

enhanced shedding of membrane-bound Hai-1/Spint1 from the neoplastic cell surface.

Given that HAI-1 shedding is reported to be mediated by metalloprotease activity (29)

and in human oral carcinoma cells MT1-MMP/MMP14 is responsible for

HAI-1/SPINT1 ectodomain shedding (30), we compared Mmp14 expression in tumor

tissue with corresponding normal mucosa in ApcMin/+ intestine and found that the

Mmp14 mRNA level was significantly increased in tumor tissue (Figure 1C). The

effect of MT1-MMP/Mmp14 on Hai-1/Spint1 shedding was then examined using the

immortalized mouse intestinal epithelial cell line MT1rev2, which was established from

Mmp14 knockout mice and carries an inducible mouse Mmp14 gene that drives

exogenous Mmp14 expression under tetracycline off conditions (28). In the MT1rev2

cells, MT1-MMP/Mmp14 expression did indeed result in enhanced Hai-1/Spint1

shedding into the culture supernatant generating 58-kDa and 42-kDa secreted forms as

previously reported (30) (Figure 1D). In accordance with these observations, mature

membrane-form Hai-1 was markedly decreased in extracts of ApcMin/+ tumors

accompanying increased amounts of 58- and 42-kDa forms and further degraded

fragments (Figure 1E). Consequently, C-terminus fragments smaller than 15 kDa were

also increased in the tumor tissue (Figure 1E).

ApcMin/+ mice deficient in intestinal Hai-1/Spint1 have shorter survival times

To test whether the loss of membrane-bound HAI-1/SPINT1 is simply an

epiphenomenon of the neoplastic transformation process in intestinal epithelial cells or

has a causal role in carcinogenesis and progression, we tested the effect of Hai-1/Spint1

defect on tumorigenesis in Apc mutant mice. By interbreeding intestine-specific

Spint1-deleted mice (Spint1flox/flox/Vil-Cre) (8) with ApcMin/+ mice, we generated

ApcMin/+ mice having a conditional deletion of the Spint1 gene in the intestinal

epithelium (ApcMin/+/Spint1flox/flox/Vil-Cre). Hai-1/Spint1 immunoreactivity was lost in

most intestinal epithelial cells in ApcMin/+/Spint1flox/flox/Vil-Cre mice, although focal

residual immunoreactivity in a small group of cells was occasionally observed (Figure

2A). The intestinal Spint1-deleted ApcMin/+ mice showed decreased body weight gain

compared to control ApcMin/+/Spint1flox/flox mice, which became apparent at 7 weeks of

age (Supplementary Figure 1), and significantly shorter survival times (p=0.0009,

log-rank test) (Figure 2B). The ApcMin/+ mice with heterozygous Spint1

(ApcMin/+/Spint1+/-) had an intermediate survival curve (Figure 2C). The major cause

of death was severe anemia probably due to increased bleeding from the intestinal tract,

which was more evident in ApcMin/+/Spint1flox/flox/Vil-Cre mice (mean hemoglobin

concentration, 5.1 g/100mL) compared to ApcMin/+/Spint1flox/flox mice (9.3 g/100mL) at

15 weeks of age (Supplementary Table 2).

Loss of intestinal Hai-1/Spint1 accelerates tumor formation in ApcMin/+ mice

We then analyzed the effect of intestinal epithelium Spint1 deletion on intestinal

tumorigenesis. Mice were sacrificed at 15 weeks of age and the number of visible

neoplastic polyps was counted. Notably, intestinal Hai-1/Spint1-deficient ApcMin/+

mice showed significantly enhanced tumor formation in the small intestine (Figure 3A,

B), a major site of tumor formation in ApcMin/+ mice, but not in the colon (Figure 3C).

The mean multiplicity (no. of tumors/mouse) in ApcMin/+/Spint1flox/flox/Vil-Cre male mice

was 95.0, which was significantly higher than that of the control ApcMin/+/Spint1flox/flox

male mice (38.5 per mouse) and the difference was particularly evident in the proximal

small intestine (Figure 3C). The sizes of tumors formed by the 15th week tended to be

larger in the intestinal Spint1-deleted mice than those in the control mice (Figure 3D).

Histologically, significant morphological differences were not observed between

Spint1-deleted tumors and control tumors (Figure 3B).

Absence of Hai-1/Spint1 enhances activation of Hgf

We compared the mRNA levels of Hai-1/Spint1 and its important target membrane

proteases, matriptase, by qRT-PCR. While matriptase (St14) mRNA levels were not

significantly altered in the intestinal tumor tissue of ApcMin/+ mouse (Figure 4A), its

subcellular localization was altered in the tumor cells, showing a decreased

membranous localization and increased intracellular immunoreactivity with punctuate

staining (Figure 4A). It should be noted that, in normal epithelium, matriptase

localization was not altered even in the absence of Hai-1/Spint1 as long as the epithelial

and villous architectures were retained.

HGF is secreted as an inactive proform (proHGF) and requires proteolytic

activation to induce c-MET-mediated signaling (11). Since all major HGF-activating

proteases (HGF activator, matriptase and hepsin) are inhibited by HAI-1/SPINT1 (11,

13), we examined the activation of proHgf in Spint1-deleted mouse intestinal tissues in

the early stage (10 weeks old) of ApcMin/+ tumorigenesis (n=4). Even at 10th week,

visible tumors are found in small intestine and the number was apparently higher in

Spint1-deleted intestine. As shown in Figure 4B, processing of Hgf was enhanced in

Spint1-deleted ApcMin/+ tumor tissue compared to control tumor tissue. Notably,

background mucosa without visible tumor (hereafter non-tumor mucosa) also showed

enhanced Hgf activation in response to Spint1 deletion. In accordance with these

observations, the immunoreactivity of phosphorylated c-Met was increased in

Spint1-deleted ApcMin/+ tumor cells compared with control cells (Figure 4B).

Wnt signaling circuit is augmented in response to intestinal deletion of Spint1 in

ApcMin/+ mice

Comprehensive gene expression profiles of intestinal mucosa and tumors from

ApcMin/+/Spint1flox/flox/Vil-Cre mice were compared with those of control

ApcMin/+/Spint1flox/flox mice using microarray, and the expression of selected genes was

further confirmed by qRT-PCR. The entire raw data set for the microarray analyses is

available at GEO. Compared with control tumors from mice of matched age,

Hai-1/Spint1-deficient ApcMin/+ tumors from 15 week old animals showed upregulation

of 22 genes that were related to the Wnt-signal pathway, including ligands, receptors,

transcription factors and downstream genes (Table 1). Among these genes, eight

(Apcdd1, Prox1, Sox17, Wif1, Fzd3, Igfbp4, Ptk7, Tcf4) were also upregulated in the

non-tumor mucosa of Spint1-deleted ApcMin/+ intestine compared with control ApcMin/+

intestine (Table 1). In addition to those 22 genes, 11 Wnt signal-related genes

including Fzd6, Wnt11, Wnt5A, Ror2 and Tcf7 (Tcf-1) were upregulated in non-tumor

mucosa in response to Spint1 deletion (Table 1). Notably, Wnt11, Apcdd1, and Sox17

mRNA levels in the Spint1-deleted non-tumor mucosa were comparable with those in

tumor tissue of control ApcMin/+ mice (Supplementary Figure 2).

Immunohistochemically, tiny foci of -catenin nuclear translocation, suggesting

preneoplastic lesion or microadenoma, were frequently observed in Spint1-deleted

non-tumor mucosa, but not in control non-tumor mucosa (Figure 4C).

Gene expression signature suggests enhanced mucosal permeability and

angiogenesis in Spint1-deleted ApcMin/+ intestine

The loss of Hai-1/Spint1 also induced upregulation of several genes possibly

related to tumor progression and angiogenesis (Supplementary Table 3). While

MT1-MMP/Mmp14 was upregulated in control ApcMin/+ tumors compared to

corresponding non-tumor mucosa (Figure 1C), it was also upregulated in

Hai-1/Spint1-deficient ApcMin/+ tumors (6.66 fold). Among these genes, up-regulation

of Flt1, Cdh13, Cdh5, and Tnfrsf12a strongly suggested enhanced angiogenesis in

Spint1-deleted tumor and non-tumor mucosa compared with control tumor and

non-tumor mucosa, respectively (Supplementary Table 3 and Figure 4D) (31-33).

Furthermore, other genes indicating increased angiogenesis, such as Vegfb, Tek, Egfl7,

Epas1, Eng, Fgfr1, Ctgf, Pf4, Hmox1, and Srf, were also upregulated in Spint1-deleted

non-tumor mucosa, although their upregulation was not apparent in tumor tissue

(Supplementary Table 4). Pml, a tumor suppressor with anti-angiogenic function (34)

was decreased (Supplementary Table 4 and Figure 4D). In fact, CD31 immunostain

revealed increased vascular density in Spint1-deleted tumors (p = 0.0005), which may

result in enhanced bleeding (Supplementary Figure 3). Cldn2 was also upregulated in

non-tumor mucosa in response to Spint1 deletion as well as in tumor tissues (Figure 4D).

Cldn2 encodes claudin-2 that enhances tight junction permeability (4), which may allow

increased permeation of genotoxic substances. In fact, several genes related to DNA

damage and/or repair were upregulated in the non-tumor mucosa of

ApcMin/+/Spint1flox/flox/Vil-Cre mice compared to that of control mice (Supplementary

Table 5).

In contrast, Spint1-deleted small intestine without Apc mutation

(Spint1flox/flox/Vil-Cre) did not show significantly upregulated expression of Cldn2, Flt1,

Apcdd1 and Sox17, compared with control Spint1flox/flox mouse intestine, and only

Tnfrsf12a showed statistically significant upregulation (Supplementary Figure 2).

Consequently, no tumor formation was observed in the intestine of Spint1flox/flox/Vil-Cre

mice with wild-type Apc gene during the observation period (15 weeks; data not

shown).

Intestinal Hai-1/Spint1 is required for resistance to chemical carcinogen-induced

colonic carcinogenesis

To test if a similar phenomenon can be reproduced under other carcinogenic

conditions without germline mutation of Apc, we examined the effect of Spint1 deletion

on chemical carcinogenesis of the colon using mice with a wild-type Apc gene.

Spint1flox/flox/Vil-Cre and control Spint1flox/flox mice received an injection of AOM

followed by administration of 1% DSS in the drinking water for five days (Figure 5A).

This carcinogenesis treatment was not efficient for the control Spint1flox/flox mice, where

only 17% of treated mice (4/23) showed tumor formation (Figure 5B). However,

defects in epithelial Hai-1/Spint1 conferred significant susceptibility to

AOM/DSS-induced tumorigenesis, as 67% (12/18) of Spint1flox/flox/Vil-Cre mice showed

tumor formation in the colon with increased multiplicity compared to the control mice

(Figure 5B). The mean size of the formed tumors was similar between the two groups

(3.73 mm and 3.58 mm for Spint1-deleted colon and control colon, respectively) (Figure

5B). The Spint1flox/flox/Vil-Cre mice that had sham treatment did not show tumor

formation. It should be noted that, similar to the ApcMin/+ mouse model described

above, the intestinal site of tumor formation was not altered by the absence of

Hai-1/Spint1 with predominant tumor formation occurring in the distal colon in both

groups (Figure 5C). While tumors in both groups showed differentiated tubular

adenocarcinoma, Spint1-deleted tumors showed less differentiated features compared to

control tumors (Figure 5D).

DISCUSSION

Using mouse knockout technology, we explored the function of the

membrane-associated serine protease inhibitor, Hai-1/Spint1, in intestinal

carcinogenesis and found that Hai-1/Spint1 has a novel role as a suppressor of

carcinogenesis in the intestinal tract. The absence of Hai-1/Spint1 in enterocytes

resulted in significantly accelerated tumor formation in ApcMin/+ mice and conferred

susceptibility to chemical carcinogenesis in the colons of wild-type Apc mice. In

accordance with these findings, cell surface Hai-1/Spint1 expression was decreased in

ApcMin/+ tumor cells in mice, and importantly, human colorectal cancer cells also

showed decreased cell surface HAI-1/SPINT1 levels compared to adjacent normal and

adenoma epithelia. Our data also suggest that the decreased amount of

membrane-associated Hai-1/Spint1 in the intestinal tumor cells may be due to

proteolytic ectodomain shedding induced by MT1-MMP/Mmp14, which implies a

novel pathway for MT1-MMP/MMP14 contribution to early neoplastic progression of

intestinal epithelium via the depletion of membrane-anchored HAI-1/SPINT1.

It should be noted that, in contrast to the mouse ApcMin/+ tumor in this study,

previous studies of human colon cancer showed decreased SPINT1 mRNA in cancer

tissues (23, 35). However, given the nearly complete loss of cell surface

HAI-1/SPINT1 immunoreactivity, the reduction levels of SPINT1 mRNA were not

sufficient in most cases: 20% and 40% reduction in well differentiated carcinomas and

overall, respectively (23, 35). In situ hybridization study also revealed a substantial

amount of SPINT1 mRNA in tumor cells that showed loss of cell surface

HAI-1/SPINT1 (25). Therefore, in human colon cancer, we suggest that both

decreased transcription and enhanced ectodomain shedding contribute in a coordinated

manner to the observed significant reduction of cell surface HAI-1/SPINT1. Recent

genome wide analyses for mutated genes in human colon cancer did not identify

relevant mutation or significant change of gene expression level of SPINT1, and only

one point mutation, which results in R390H (R406H in HAI-1B, a common splicing

variant), was found (36). Collectively, post-translational enhanced ectodomain

shedding might be an important mechanism underlying the loss of cell surface

HAI-1/SPINT1 in tumor cells.

Multiple mechanisms could potentially contribute to the suppressive effect of the

membrane-associated Hai-1/Spint1 on intestinal tumorigenicity, but so far the evidence

suggests that its roles for epithelial integrity to maintain the barrier function and

regulation of Hgf processing are responsible. A previous study indicated that,

although mice lacking intestinal Hai-1/Spint1 showed normal development and growth,

intestinal permeability was increased and these mice had enhanced susceptibility to

DSS-induced colitis (8). In this study, Hai-1/Spint1 loss induced acceleration of tumor

formation in ApcMin/+ mice or when mice received a carcinogenic insult, but not in mice

with wild-type Apc or untreated mice. Therefore, the pivotal function of Hai-1/Spint1

likely emerges when epithelial cells encounter pathological stimuli and/or cellular stress

due to genetic abnormalities. Under adverse cellular conditions, the loss of

Hai-1/Spint1 may result in increased epithelial permeability that could amplify these

pathological stimuli and accelerate accumulation of genetic abnormalities.

Upregulation of claudin-2 expression would further enhance epithelial permeability in

Spint1-deleted ApcMin/+ mice, as this particular claudin correlates with epithelial

leakiness (4, 5). Moreover, claudin-2 is reported to be involved in colon tumorigenesis

(37). Enhanced activation of Hgf and its specific receptor c-Met may also be an

important mechanism for tumorigenesis enhanced by Hai-1/Spint1 loss, as

HAI-1/SPINT1 is a potent inhibitor of major proHGF activating proteases, HGF

activator, matriptase, and hepsin (6, 13, 16) and important roles of HGF/c-MET in

cancer progression have been established (38). The enhanced Hgf activation may also

be involved, at least partly, in enhanced angiogenesis in ApcMin/+ tumors (38).

The cross talk between HGF/c-MET signaling and WNT/-catenin signaling has

been reported. The activated HGF secreted by stromal myofibroblasts reportedly

activates -catenin-dependent transcription and subsequent colon cancer stem cell

clonogenicity, and also restores the cancer stem cell phenotype in more differentiated

tumor cells (39). HGF-induced activation of WNT pathway via transcriptional

activation of lymphoid enhancer binding factor 1 (LEF1) was also reported in human

cancer cells (40). Indeed, comprehensive gene expression profiling revealed that the

Spint1-deleted ApcMin/+ intestine showed significant upregulation of proteins involved in

the Wnt-signaling circuit and enhanced angiogenesis even in non-tumor mucosa, which

would provide a cancer-promoting microenvironment. T-cell factor (Tcf) 4, part of the

Tcf4-Lef1 complex, was upregulated in Spint1-deleted mucosa. In addition, recent

study suggested that Tcf7 (also known as Tcf-1) and Lef1 are obligate partners in the

oncogenic activities of -catenin in Apc mutant mice (41), so it is of interest that Tcf7

gene expression was also upregulated in Spint1-deleted mucosa. In accordance with

these findings, Sox17 and Apcdd1 genes were markedly upregulated not only in tumor

tissue but also in the Hai-1/Spint1-deficient non-tumor mucosa. Sox17 is induced by

Wnt signal activation in the early stage of gastrointestinal tumorigenesis (42) and

Apcdd1 is also regulated by the -catenin/Tcf complex with its elevated expression

contributing to colorectal carcinogenesis (43). As these alterations in gene expression

in the non-tumor mucosa were not observed in the Spint1-deleted intestine with

wild-type Apc, microadenomas intermingling with normal epithelium may contribute to

the upregulation of these genes. However, the degrees of upregulation of some genes,

such as Apcdd1 and Sox17, in Spint1-deleted non-tumor mucosa were comparable with

tumor tissue itself from control ApcMin/+ mice, indicating that Spint1 deletion amplified

Wnt signaling. Taken altogether, the function of intestinal Hai-1/Spint1 as a tumor

suppressor retards the formation of a tumor-promoting microenvironment under

cancer-prone epithelial conditions and does not act as a gatekeeper in carcinogenesis.

This notion is supported by the fact that enhanced tumor formation was observed only

in its proper site in each of the mouse models examined in this study: small intestine in

ApcMin/+ mice or distal large intestine in the AOM/DSS carcinogenesis model.

Matriptase is one of the most important cognate proteases of HAI-1/SPINT1

(14-17) and is critically required to maintain intestinal epithelial integrity (3-5).

Therefore, deregulated matriptase activity may be involved in the phenotypes observed

here for Hai-1/Spint1 deficiency. In fact, the ratio of matriptase to HAI-1/SPINT1 is

higher in colorectal cancers compared to normal tissue (44) and cancer cells show

increased matriptase activity (45). However, there is an apparent paradox for the

intestinal tumorigenesis in the matriptase:Hai-1/Spint1 balance. Matriptase

hypomorphic mice showed enhanced susceptibility to DSS colitis and increased

claudin-2 expression in intestinal epithelial cells (4). Conditional deletion of the

mouse matriptase gene (St14) in the intestinal tract impairs colonic mucosal integrity

and induces colonic cancer development (3, 46). Therefore, similar to Hai-1/Spint1,

matriptase is a tumor suppressor in the intestinal tract. We hypothesize that

membranous Hai-1/Spint1 may be critical for “normal” matriptase function that is

required for epithelial integrity and loss of Hai-1/Spint1 may cause abnormal

subcellular localization or enhanced release of matriptase under adverse cellular

conditions. In fact, HAI-1/SPINT1 is required for intracellular matriptase trafficking

(15, 16, 47), and in tumor cells, loss of membrane-associated HAI-1/SPINT1 resulted in

decreased membrane-associated matriptase and increased pericellular matriptase

activity (27, 30) that might activate pericellular molecules involved in tumor

progression such as proHGF. In the current study, intestinal tumor cells with decreased

membranous Hai-1/Spint1 showed abnormal matriptase subcellular localization.

However, in non-neoplastic epithelial cells, the cell surface localization pattern of

matriptase was preserved in Spint1-deleted cells as long as the epithelium retained a

normal histological architecture. In this regard, Hai-2/Spint2 is also expressed in the

intestinal epithelium and may compensate for the loss of Hai-1/Spint1 in normal

epithelial cells (8).

A potential limitation of this study is the question whether total absence of

Hai-1/Spint1 in the Spint1-delete intestinal tumor cells exactly represents the

pathobiology of the tumor cells with ectodomain shedding-induced loss of cell surface

Hai-1/Spint1. Although known functional domain of HAI-1/SPINT1 is extracellular

N-terminal Kunitz domain and no function has been reported for C-terminal

intracytoplasmic domain, remnant C-terminus of HAI-1/SPINT1 after ectodomain

shedding may acquire unexpected biological functions. This possibility should be

clarified in a future study. Another important issue in this study that requires further

clarification is whether a novel pro-tumorigenic protease regulated by Hai-1/Spint1 may

exist in intestinal epithelial cells. Deregulated activity of this putative protease in the

absence of Hai-1/Spint1 may contribute to accelerated tumorigenesis. In this regard,

several Hai-1/Spint1-sensitive membrane serine proteases, such as TMPRSS13,

TMPRSS4, hepsin, and prostasin are also expressed in the intestinal tract (8).

Prostasin activates epithelial sodium channel and protease-activated receptor 2 (6),

which may be involved in intestinal tumorigenesis (48). While the biological function

of TMPRSS4 is poorly understood, this TTSP does induce an invasive phenotype in

cancer cells (20, 49).

In conclusion, our data suggest that the epithelial cell surface protease-inhibitor

interaction may impact mucosal carcinogenesis and the loss of membranous

HAI-1/SPINT1 in epithelial cells confers susceptibility to tumorigenesis in the intestinal

tract. Further analysis of the molecular mechanisms that underlie enhanced intestinal

tumor formation in the Spint1-deleted mouse carcinogenesis models described in this

study may reveal novel targets for therapeutic intervention.

Acknowledgement: The authors are grateful to Ms. Yumiko Nomura and Ms. Ai

Kanemaru for preparation of histological specimens.

Grant support: This work was supported by Grant-in-Aid for Scientific Research no.

24390099 (H.K.) and no. 23790250 (M.K.) from the Ministry of Education, Science,

Sports and Culture, Japan.

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Figure legends

Figure 1. Decreased membranous HAI-1/SPINT1 in intestinal tumor cells and its

shedding by MT1-MMP/MMP14. A, immunohistochemistry of HAI-1/SPINT1 in

human colon adenomas and carcinomas. Serial sections were stained with H&E and

anti-HAI-1 antibody (bar, 100 μm). B, immunohistochemistry of Hai-1/Spint1 in

intestinal tumors of ApcMin+ mice (bar, 50 μm). Data for qRT-PCR of Spint1 mRNA

levels in tumor and corresponding non-tumor mucosa tissue (n = 3) are also indicated.

C, enhanced MT1-MMP/Mmp14 expression in mouse intestinal tumor compared to the

corresponding normal mucosa. Data for qRT-PCR of Mmp14 mRNA levels (n = 5) are

also indicated. D, doxycycline (Dox) off-induced expression of MT1-MMP/Mmp14

(RT-PCR) and enhanced shedding of Hai-1/Spint1 by MT1-MMP/Mmp14

(immunoblot) in MT1rev2 cells. sHai-1, secreted-form Hai-1. Data in bar graphs are

mean ± SD. *, p = 0.008, Mann-Whitney U test. E, decreased mature membrane form

and increased cleaved fragments of Hai-1/Spint1 in ApcMin/+ tumor tissues. Arrow and

arrow head indicate fragments compatible with 58-kDa and 42-kDa sHAI-1,

respectively, observed in MT1-MMP/Mmp14-expressing MT1rev2 supernatant, and

asterisk indicates ~15-kDa C-terminus fragments of Hai-1/Spint1.

Figure 2. Generation of intestinal Hai-1/Spint1-deficient ApcMin/+ mice and their

survival curves. A, Hai-1/Spint1 immunostaining of small intestine from control

(ApcMin/+/Spint1flox/flox) and intestinal epithelium-specific Spint1-knockout mice

(ApcMin/+/Spint1flox/flox/Vil-Cre). Focal residual Hai-1/Spint1 immunoreactivity is

indicated by arrows. Bars, 100 (upper) and 20 (lower panel) μm. RT-PCR for Spint1

and matriptase mRNAs in proximal small intestine (pSI), distal small intestine (dSI),

proximal large intestine (pLI) and distal large intestine (dLI) are also shown. B,

Kaplan-Meyer survival curves of ApcMin/+/Spint1flox/flox/Vil-Cre (n = 12, all males) and

control mice (ApcMin/+/Spint1flox/flox; n = 10, all males). The Spint1 mutant mice

showed shorter survival times (p = 0.0009, log-rank test). C, survival curves of

ApcMin/+/Spint1flox/flox/Vil-Cre (n = 20, 12 males and 8 females), ApcMin/+/Spint1+/-

(Spint1 heterozygous; n = 5, one male and 4 females) and ApcMin/+/Spint1+/+ (wild-type

Spint1; n = 20, 8 males and 12 females) mice. ApcMin/+/Spint1flox/flox/Vil-Cre mice

showed shorter survival times compared with ApcMin/+/Spint1+/- (p = 0.0008, log-rank

test) and ApcMin/+/Spint1+/+ (p < 0.0001, log-rank test) mice.

Figure 3. Enhanced tumor formation in the small intestine of ApcMin/+ mice in

response to Spint1 deletion. A, Macroscopic appearance of small intestines from

ApcMin/+/Spint1flox/flox (cont) and ApcMin/+/Spint1flox/flox/Vil-Cre (KO) mice (bar, 1 cm).

B, microscopic findings of intestinal tumors from ApcMin/+/Spint1flox/flox (cont) and

ApcMin/+/Spint1flox/flox/Vil-Cre (KO) mice. In whole intestine sections (upper two

panels, H&E), enhanced tumor formation is evident in KO mice (bar, 1 mm). Higher

magnification photos of control and KO tumors (lower left two panels, H&E) indicate

severely dysplastic tubules of similar histology (bar, 20 μm). Immunohistochemistry

for Hai-1/Spint1 (lower right three panels) shows absence of Hai-1/Spint1 in both

normal epithelium and tumor cells of KO intestine. In control intestine, neoplastic

epithelium (T) showed decreased immunoreactivity of cell surface Hai-1/Spint1. Bars

represent 100 μm and 20 μm for low and high magnification photos, respectively. C,

number of visible polyps in the intestines (mean ± SEM). *, p = 0.0001; **, p =

0.0084 (Mann-Whitney U test). D, comparison of polyp size scores. The box shows

the interquartile range, the whiskers the largest and smallest observed scores that are <

1.5 box length from the end of the box, and the median is indicated by a bold vertical

line. *, P = 0.0018 (Mann-Whitney U test).

Figure 4. Enhanced Hgf processing and upregulation of Wnt signal-related genes. A,

qRT-PCR for Spint1 and matriptase (St14) and matriptase localization

(immunofluorescence) in small intestine. Non-tumor mucosa (non-T) and tumor (T)

from small intestine of ApcMin/+/Spint1flox/flox (C, control) or

ApcMin/+/Spint1flox/flox/Vil-Cre (KO, Spint1 deletion) mice were analyzed. qRT-PCR

data are means ± SD (*, p < 0.0001; NS, not significant; n = 3). Green fluorescence,

matriptase; blue, nuclei. N, non-neoplastic epithelium; T, neoplastic epithelium. Bar,

20 μm. B, processing of proHgf in non-tumor mucosa (upper panel) and tumor tissues

(lower panel) (10 weeks old). Representative immunoblot photos and bar graph data

of relative processing rate (n = 4) are shown. *, p < 0.05. Immunostaining photos of

phosphorylated c-Met in intestinal neoplastic lesions are also shown. Bar, 20 μm. C,

immunostaining of -catenin (b-cat) of non-tumor mucosa (upper panels) and tumor

mucosa (lower panels). Arrows indicate nuclear translocation of -catenin in epithelial

cells of non-tumor mucosa from Spint1-deleted intestine and enlarged image is also

shown as inset. In the last panel, phosphorylated c-Met (pMet) was also

immunostained using a serial section. Bar, 20 μm. D, RT-PCR of angiogenesis

related genes and Cldn2 and qRT-PCR analysis of Cldn2 expression. *, p = 0.0008.

P values were analyzed by Mann-Whitney U test.

Figure 5. Effect of Spint1 deletion on AOM/DSS-induced colon carcinogenesis. A,

AOM/DSS treatment protocol. B, tumor multiplicity and size. The number of visible

polyps/mouse was counted and the maximum diameter of each polyp measured. cont,

Spint1flox/flox; KO, Spint1flox/flox/Vil-Cre. Bold vertical line indicates the mean value.

*, p = 0.0002 (Mann-Whitney U test). C, representative photo of macroscopic

findings of distal colon tissues from Spint1flox/flox (cont) and Spint1flox/flox/Vil-Cre (KO)

mice. Bar, 1 cm. D, histology (H&E) and Hai-1/Spint1 immunostaining of colon

tumors. Photos in upper panel are tumor formed in Spint1flox/flox (cont) mouse,

showing decreased Hai-1/Spint1 immunoreactivity in tumor cells. Dotted line

indicates boundary between tumor (T) and non-neoplastic epithelium (N). Middle

panel indicates low magnification histology (H&E) of Spint1flox/flox/Vil-Cre (KO) mouse

tumor that lacks Hai-1/Spint1 expression (inset). Photos in the lowest panel indicate

comparative histology of cont and KO tumors. Tumor cells in KO mouse show

decreased mucin production. Bar, 50 μm.

Table 1. Effects of Spint1 deletion on Wnt signaling-related gene expression

Up- or down-regulated (> 2.0 fold) genes in response to Spint1 deletion Fold change Annotation & Gene symbol (Entrez Gene ID) Tumor Non-tumor Adenomatosis polyposis coli down-regulated 1, Apcdd1 (494504) 6.59 4.63 -Catenin, Ctnnb1 (12387) 5.08 NA LIM domain binding 1, Ldb1 (16825) 4.37 NA Prospero-related homeobox 1, Prox1 (19130) 4.16 2.93 Frizzled homolog 2, Fzd2 (57265) 4.09 NA Protein inhibitor of activated STAT4, Pias4 (59004) 3.39 NA Casein kinase 1 delta, Csnk1d (104318) 3.32 NA F-box and WD-40 domain protein 2, Fbxw2 (30050) 3.22 NA SRY-box containing gene 17, Sox17 (20671) 3.21 10.28 Wnt inhibitory factor 1, Wif1 (24117) 3.06 3.33 Casein kinase 1 gamma 2, Csnk1g2 (103236) 2.98 NA Casein kinase 2 alpha 1, Csnk2a1 (12995) 2.87 - 2.11 Frizzled homolog 3, Fzd3 (14365) 2.80 3.66 Low density lipoprotein receptor-related protein 6, Lrp6 (16974) 2.60 NA Methyl-CpG binding domain protein 2, Mbd2 (17191) 2.42 NA Insulin-like growth factor binding protein 4, Igfbp4 (16010) 2.38 4.97 Protein phosphatase 1A, alpha isoform, Ppm1a (19042) 2.36 - 2.01 Protein tyrosine kinase 7, Ptk7 (71461) 2.35 14.34 Casein kinase 1 alpha 1, Csnk1a1 (93687) 2.26 NA Low density lipoprotein receptor-related protein 5, Lrp5 (16973) 2.09 NA Transcription factor 4, Tcf4 (21413) 2.06 2.11 SUMO/sentrin specific peptidase 2, Senp2 (75826) 2.01 NA Frizzled homolog 6, Fzd6 (14368) NA 4.28 Naked cuticle 1 homolog, Nkd1 (93960) NA 4.16 Dickkopf homolog 3, Dkk3 (50781) NA 3.77 Wingless-type MMTV integration site 11, Wnt11 (22411) NA 3.55 Receptor tyrosine kinase-like orphan receptor 2, Ror2 (26564) NA 2.98 Wingless-type MMTV integration site 5A, Wnt5A (22418) NA 2.49 Casein kinase 1 epsilon, Csnk1e (27373) NA 2.48 SRY-box containing gene 4, Sox4 (20677) NA 2.46 Transcription factor 7, Tcf7 (also known as Tcf-1) (21414) NA 2.41 Microtubule-actin crosslinking factor 1, Macf1 (11426) NA 2.21 Lymphoid enhancer binding factor 1, Lef1 (16842) NA 2.02 Transducin-like enhancer of split 3, Tle3 (21887) NA - 2.28

Hoshiko et al, Figure 2

A ApcMin/+Spint1flox/flox ApcMin/+Spint1flox/floxVil-Cre

ApcMin/+ Spint1flox/flox

pSI dSI pLI dLI Hai-1 matriptase 㽪-actin

ApcMin/+ Spint1flox/flox Vil-Cre

pSI dSI pLI dLI Hai-1 matriptase 㽪-actin

B C

100 100 ApcMin/+ +/+ ApcMin/+Spint1flox/flox Spint1

80 80

60 60

40 40 ApcMin/+Spint1+/- Survival rate (%) Survival rate (%)

20 20 ApcMin/+Spint1flox/flox Vil-Cre ApcMin/+Spint1flox/flox Vil-Cre

0 0 0 525351510 20 30 0 525351510 20 30 Age (weeks) Age (weeks)

Hepatocyte growth factor activator inhibitor type 1 (Hai-1/Spint1) is a suppressor of intestinal tumorigenesis

Shinri Hoshiko, Makiko Kawaguchi, Tsuyoshi Fukushima, et al.

Cancer Res Published OnlineFirst February 27, 2013.

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