stress response protein cirp links inflammation and...

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Stress response protein Cirp links inflammation and tumorigenesis in

colitis-associated cancer

Running title: Cirp and inflammatory bowel disease

Toshiharu Sakurai1*, Hiroshi Kashida1, Tomohiro Watanabe2, Satoru Hagiwara1,

Tsunekazu Mizushima4, Hideki Iijima5, Naoshi Nishida1, Hiroaki Higashitsuji3, Jun

Fujita3, Masatoshi Kudo1

1Department of Gastroenterology and Hepatology, Kinki University Faculty of

Medicine, Osaka-Sayama, 2Center for Innovation in Immunoregulative Technology and

Therapeutics, 3Department of Clinical Molecular Biology, Graduate School of Medicine,

Kyoto University, Kyoto, Departments of 4Surgery and 5Gastroenterology and

Hepatology, Osaka University, Osaka, Japan

Abbreviations: AOM, azoxymethane; BM, bone marrow; BMT, bone marrow

transplantation; Cirp; cold-inducible RNA-binding protein; CD, Crohn’s disease; CAC,

colitis-associated cancer; DSS, dextran sodium sulfate; ERK, extracellular

signal-regulated protein kinase; IBD, inflammatory bowel disease; LP, lamina propria;

MAPK, mitogen-activated protein kinase; UC, ulcerative colitis; WT, wild type.

* To whom correspondence may be addressed. E-mail: [email protected]

on March 22, 2019. © 2014 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 3, 2014; DOI: 10.1158/0008-5472.CAN-14-0471

http://cancerres.aacrjournals.org/

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Department of Gastroenterology and Hepatology, Kinki University Faculty of Medicine,

377-2 Ohno-Higashi, Osaka-Sayama, Osaka, 589-8511, Japan

Tel.: +81 72 366 0221(ext. 3525); Fax: +81 72 367 2880

Conflict of interest: The authors have declared that no conflict of interest exists.

Keywords: Sox2, Bcl-2, IL-23, stem cell, colitic cancer, therapy resistance




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Abstract

Colitis-associated cancer (CAC) is caused by chronic intestinal inflammation and

reported to be associated with refractory inflammatory bowel disease (IBD). Defective

apoptosis of inflammatory cell populations seems to be a relevant pathogenetic

mechanism in refractory IBD. We assessed the involvement of stress response protein

cold-inducible RNA-binding protein (Cirp) in the development of intestinal

inflammation and CAC. In the colonic mucosa of ulcerative colitis (UC) patients,

expression of Cirp correlated significantly with the expression of TNF-α, IL-23/IL-17,

anti-apoptotic proteins Bcl-2 and Bcl-xL and stem cell markers such as Sox2, Bmi1 and

Lgr5. The expression of Cirp and Sox2 was enhanced in the colonic mucosae of

refractory UC, suggesting that Cirp expression might be related to increased cancer risk.

In human CAC specimens, inflammatory cells expressed Cirp protein. Cirp-/- mice

given dextran sodium sulfate exhibited decreased susceptibility to colonic inflammation

through decreased expression of TNF-α, IL-23, Bcl-2 and Bcl-xL in colonic lamina

propria cells compared with similarly treated wild type (WT) mice. In the murine CAC

model, Cirp deficiency decreased the expression of TNF-α, IL-23/IL-17, Bcl-2, Bcl-xL

and Sox2 and the number of Dclk1+ cells, leading to attenuated tumorigenic potential.

Transplantation of Cirp-/- bone marrow into WT mice reduced tumorigenesis, indicating

the importance of Cirp in hematopoietic cells. Cirp promotes the development of

intestinal inflammation and colorectal tumors through regulating apoptosis and

production of TNF-α and IL-23 in inflammatory cells.




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Introduction

The inflammatory bowel diseases (IBD); ulcerative colitis (UC) and Crohn’s disease

(CD) are thought to result from aberrant activation of the intestinal mucosal immune

system (1). Although the pathogenesis of IBD remains unclear, a number of studies

have suggested the involvement of abnormal apoptosis in intestinal epithelial cells,

resulting from increased production of cytokines, such as tumor necrosis factor (TNF),

interleukins (ILs), and interferons (2). TNF-α is a key mediator of inflammation in IBD

and has been the primary target of biologic therapies (3). This cytokine induces

inflammation by promoting the production of IL-1� and IL-6, expression of adhesion

molecules, proliferation of fibroblasts, activation of procoagulant factors, and

cytotoxicity of the acute phase response (4). The IL-23/Th17 (T-helper IL-17-producing

cell) pathway has been identified to play a critical role in IBD. IL-23 has been shown to

promote the expansion of a distinct lineage of Th17 cells that are characterized by

production of a number of specific cytokines not produced by Th1 or Th2 cells,

including IL-17A, IL-17F, IL-21 and IL-22 (5). IL-23/IL-17 signaling enhances the

immunosuppressive activity of regulatory T cells and reduces CD8+ cells in tumor,

leading to enhanced tumor initiation and promotion (6, 7). Recently, a study has

suggested that colorectal cancer tissue-derived Foxp3+ IL-17+ cells have the capacity to

induce cancer-initiating cells in vitro (8). The most conspicuous link between

inflammation and colon cancer is seen in patients with IBD (9), and development of

colorectal cancer is one of the most serious complications of IBD, which is also referred

to as colitis-associated cancer (CAC) (10). Thus, it is of great importance to improve

our understanding of the molecular link between chronic inflammation and CAC in




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order to identify a target molecule with therapeutic potential for the treatment of IBD

and prevention of CAC.

It is widely accepted that most tumors harbor cancer stem cells, which are crucial

for a tumor’s evolutionary capability. Cancer stem cells resemble normal stem cells in

their capacity to self-renew and continuously replenish tumor progeny (11, 12). The G

protein-coupled receptor Lgr5 and the polycomb group protein Bmi1 are two recently

described molecular markers of the self-renewing multipotent adult stem cell

populations residing in intestinal crypts that mediate regeneration of the intestinal

epithelium (13, 14). Pluripotency-associated transcription factors like Sox2 are known

to regulate cellular identity in embryonic stem cells. Sox2 expression specifically

increased the numbers of stem cells and repressed Cdx2, a master regulator of

endodermal identity. In vivo studies demonstrated that Sox21, another member of the

SoxB gene family, was a specific, immediate, and cell-autonomous target of Sox2 in

intestinal stem cells (15). Sox2 participates in the reprogramming of adult somatic cells

to a pluripotent stem cell state and is implicated in tumorigenesis in various organs (16).

Cold-inducible RNA-binding protein (Cirp, also called Cirbp or hnRNP A18) was

originally identified in the testis as the first mammalian cold shock protein (17) and is

suggested to mediate the preservation of neural stem cells (18). Cirp is induced by

cellular stresses such as UV irradiation and hypoxia (19-21). In response to the stress,

Cirp, which migrates from the nucleus to the cytoplasm, affects post-transcription

expression of its target mRNAs (22-24) and functions as a damage-associated molecular

pattern molecule that promotes inflammatory responses when present extracellularly

(25). Cirp also affects cell growth and cell death induced by TNF-α or genotoxic stress

(26, 27). However, the involvement of Cirp in colitis and CAC is not well understood.




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Here we examined whether Cirp plays a role in inflammatory immune responses

and tumorigenesis in the gut by using a murine CAC model of Cirp-deficient (Cirp-/-)

mice and found that Cirp promoted colitis and colorectal tumorigenesis by inhibiting

apoptosis and increasing TNF-α and IL-23 production in inflammatory cells. In UC

patients, refractory inflammation is associated with increased Cirp expression in the

colonic mucosa, which would increase the risk for CAC. This study represents the first

report of the functional link between Cirp and intestinal tumorigenesis.

Materials and Methods

Human tissue samples

In total, 236 colonic mucosa specimens were obtained by endoscopy or surgery

from UC patients including 67 cases of refractory UC, 98 cases of non-refractory active

UC, and 20 cases in remission, as well as 21 colonic mucosa of CD patients and 30

normal colonic mucosa specimens from controls without IBD. Refractory UC was

defined according to endoscopic criteria and categorized as being active for more than 6

months. Active inflammation was defined as Mayo endoscopic score >2. CAC

specimens were obtained from 10 patients who had undergone colorectal resection. The

clinical study protocol conformed to the ethical guidelines of the 1975 Declaration of

Helsinki and was approved by the relevant institutional review boards.

Mice and treatment

Cirp-/- mice showing neither gross abnormality nor colonic inflammation were used

as a murine CAC model. The generation of Cirp-/- mice has been described previously




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(28). Sex- and age-matched C57BL/6 wild type (WT) and Cirp-/- mice (8–12 weeks old)

received 2.5% (w/v) dextran sodium sulfate (DSS; molecular weight, 36,000–50,000

kDa; MP Biomedicals, Solon, OH) in drinking water. Mice were i.p. injected with 20

mg/kg anti-TNF-α antibody (#16-7423, eBioscience, San Jose, CA) or an IgG isotype

control before DSS treatment.

Isolation of lamina propria (LP) cells was performed as described previously (29).

The isolated cells were sorted using immunomagnetic beads coated with monoclonal

antibodies against CD11b (MACS Beads, Miltenyi Biotec, Bergisch Gladbach,

Germany) with the help of a separation column and a magnetic separator from the same

company in accordance with the manufacturer’s recommendations for isolating murine

macrophages.

As the protocol for the murine CAC model, mice were intraperitoneally injected

with 12.5 mg/kg azoxymethane (AOM; Sigma-Aldrich, St. Louis, MO). After 5 days,

2.0% dextran sodium sulfate (DSS) was included in the drinking water for 5 days,

followed by 16 days of regular water. This cycle was repeated three times. Then, 1.5%

DSS was included in the drinking water for 4 days, followed by 7 days of regular water.

Upon sacrifice, the colon was excised from the ileocecal junction to the anus, cut open

longitudinally, and prepared for histological evaluation. Colons were assessed

macroscopically for polyps under a dissecting microscope.

Bone marrow transplantation (BMT) experiments were performed as previously

described, with slight modifications (30). Bone marrow (BM) from the tibia and femur

was washed twice in Hanks balanced salt solution, and 107 bone marrow cells were

injected into the tail vein of lethally irradiated (11 Gy) recipient mice. Eight weeks

post-transplantation, the mice were subjected to the murine CAC protocol. BM cells




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were grown in culture dishes in the presence of M-CSF (10 ng/ml) and then

differentiated to BM-derived macrophages in 10 days. All animal procedures were

performed according to approved protocols and in accordance with the

recommendations for the proper care and use of laboratory animals. The animal study

protocol was approved by the Medical Ethics Committee of Kinki University School of

Medicine.

Colonic injury scoring

Excised colons were rolled up and fixed in 10% formaldehyde, embedded in

paraffin, and stained with hematoxylin and eosin (H&E). The degree of colonic injury

was assessed by histologic scoring as described previously (31), with minor

modifications. The protocol is described in detail in Supplementary Materials and

Methods.

Biochemical and immunochemical analyses

Real-time qPCR, immunoblotting, and immunohistochemistry were previously

described (32). Primer sequences are given in Supplementary Materials and Methods.

The following antibodies were used: anti-actin, anti-DCAMLK1 (Dclk1)

(Sigma-Aldrich); anti-Bcl-2, anti-phospho-IκBα, anti- IκBα, anti-phospho-ERK,

anti-ERK, anti-Sox2, anti-E-cadherin, anti-PCNA (Cell Signaling, Danvers, MA); and

anti-F4/80 (eBioscience). Generation of anti-Cirp polyclonal antibody was previously

described (28). Immunohistochemistry was performed using ImmPRESSTM reagents

(Vector Laboratory, Burlingame, CA) according to the manufacturer’s recommendations.

Immunofluorescent TUNEL staining was performed to measure apoptosis in




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paraffin-embedded sections using the In Situ Apoptosis Detection Kit as described by

the manufacturer (Takara, Tokyo, Japan). Nuclei were stained with 4,

6’diamidino-2-phenylindole to count the total cells per crypt. A minimum of 10 crypts

were counted per section.

Statistical analysis

Differences were analyzed using Student’s t-test. To compare variables of more than

2 conditions, analysis of variance (ANOVO) with post-hoc Tukey-Kramer honestly

significant difference (HSD) multiple comparison was applied. The relationship

between the expression of several genes was analyzed by Spearman’s rank correlation

test. P values <0.05 were considered significant.

Results

Correlation of Cirp expression with TNF-α, IL-23/IL-17, Bcl-2 and stem cell

marker expression in patients with IBD

Cirp expression correlated weakly but significantly with TNF-α with a linear

coefficient of 0.26 in the colonic mucosa of UC patients (Fig. S1A). In CD patients,

Cirp expression did not significantly correlate with TNF-α (data not shown) probably

because the majority of CD patients enrolled in this study had undergone anti-TNF-α

therapy. IL-23p19 is the specific subunit of IL-23, a positive regulator of Th17 and other

IL-17-producing cells (5). A significant correlation was found between Cirp and IL-17A

or IL-23p19 mRNA expression in UC patients (Fig. S1B and S1C) and in CD patients

(Fig. S2A).




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Defective apoptosis of inflammatory cell populations regulated by Bcl-2 seems to be

a relevant pathogenetic mechanism in IBD (33, 34). There was a significant correlation

between Cirp and Bcl-2 expression with a linear coefficient of 0.76 in UC patients and

with a linear coefficient of 0.60 in CD patients (Fig. 1A and Fig. S2B). Expression of

Bcl-xL, another anti-apoptotic protein, was significantly correlated with that of Cirp

with a linear coefficient of 0.60 in UC patients (Fig. S1D) and with a linear coefficient

of 0.85 in CD patients (Fig. S2C).

Stem cells, characterized by their ability to self-renew indefinitely and produce

progeny capable of repopulating tissue-specific lineages, are critical for maintaining

normal tissue homeostasis (35). Cirp is suggested to mediate the preservation of neural

stem cells (18). Cirp expression correlated with Sox2, Bmi1, Lgr5 and Dclk1 levels

with linear coefficients of 0.62, 0.45, 0.42 and 0.25, respectively in UC patients (Fig.

1B-D and Fig. S1E) and correlated with Sox2 with a linear coefficient of 0.63 in CD

patients (Fig. S2D). Cirp might be involved in regulation of intestinal inflammation and

homeostasis maintenance in IBD patients.

Increased Cirp expression in the colonic mucosa of patients with refractory UC

We next explored whether an association exists between Cirp expression and the

clinical status of UC patients. Refractory and non-refractory active UC could not be

distinguished by endoscopic findings (Fig. 2A). Cirp expression levels were specifically

increased in patients with refractory UC associated with long-term inflammation, whilst

similar expression levels of Cirp were found between normal colonic mucosa and the

mucosa of patients with non-refractory active UC (Fig. 2B). Similarly, increased Sox2

expression levels were found in the colonic mucosa of refractory IBD patients (Fig. 2C).




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Immunohistochemistry showed that Sox2 was expressed in the mesenchyme and Dclk1

was expressed in the crypt of refractory UC patients (Fig. S3A and S3D). In contrast,

increased TNF-α expression was found in the colonic mucosa of both refractory and

non-refractory active UC (Fig. 2D). Immunohistochemistry was performed to identify

the cells expressing Cirp in the human intestine, and inflammatory cells were found to

express more Cirp protein than epithelial cells, whose expression pattern was similar in

controls and patients with UC. In chronically inflamed mucosa, Cirp expression was

enhanced in inflammatory cells (Fig. 2E and Fig. S3B). Inflammatory cells

preferentially but not exclusively expressed Cirp protein also in human CAC cases (Fig.

S3C).

Cirp-/- mice challenged with DSS have decreased susceptibility to inflammation

DSS-induced colitis is a murine model resembling human UC. Experimental colitis

was induced by treating mice with 2.5% DSS. Histological analysis revealed

substantially less epithelial damage and disruption of crypt architecture in Cirp-/- mice

than in WT mice (Fig. 3A), and inflammatory cell infiltration into the colon was less in

Cirp-/- mice (Fig. 3B). Immunohistochemically assessed macrophage infiltration was

smaller in Cirp-/- than in WT mice after DSS administration (Fig. 3C), and the epithelial

injury score was significantly smaller in the Cirp-/- mice than in the controls (Fig. 3D).

Next, we compared apoptosis induction in DSS-treated WT and Cirp-/- mice. Apoptosis

detected by TUNEL staining was observed in DSS-treated mice, primarily in the colonic

crypts (Fig. 3E), but was blocked by 50% in the Cirp-/- mice (Fig. 3F). Examination of

the colonic lysates from DSS-treated WT and Cirp-/- mice showed that the Cirp presence

increased PCNA expression in the colon (Fig. S4A).




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The associated immune response was investigated by analyzing colonic cytokine

levels. Colonic tissue from Cirp-/- mice showed a smaller immune response with lower

levels of pro-inflammatory cytokine TNF-α and IL-23 than that of WT mice (Fig.

4A-C), which is consistent with the data in humans (Fig. 1 and Fig.S1). TNF-α

expression was upregulated in non-refractory active UC whilst Cirp expression was not

in these patients (Fig. 2B and 2D). This is probably because TNF-α is induced in both a

Cirp-dependent and -independent manners in the colon. There was no significant

difference in IL-1β, IL-10 and IL-21 (Fig. 4B and Fig. S4B). To explore the

mechanisms of the effects of Cirp on TNF-α production, we sought to confirm these

findings in vitro. In lamina propria (LP) cells isolated from DSS-treated colons of

Cirp-deficient mice, expression of TNF-α and IL-23 were decreased compared with

those of WT mice (Fig. 4D). TNF-α is produced chiefly by activated macrophages,

although it can be produced by many other cell types as lymphocytes and NK cells (36),

so we next isolated macrophages from DSS-treated colons. TNF-α mRNA expression

was significantly reduced in Cirp-deficient macrophages (Fig. 4E). Macrophages

derived from WT bone marrow (BM) exhibited marked upregulation of TNF-α mRNA

relative to those derived from Cirp-/- BM (Fig. S4C). Cirp has been reported to activate

NF-κB (25, 27). Consistently, the presence of Cirp increased IκBα phosphorylation in

bone marrow (BM)-derived macrophages (Fig S4D). Treatment with anti-TNF-α

antibody reduced inflammatory cell infiltration in WT mice but not in Cirp-/- mice (Fig.

4F). These data indicate that Cirp in inflammatory cells augments the inflammatory

response by producing cytokines such as TNF-α and IL-23. In addition, Bcl-2 and

Bcl-xL mRNA expression was significantly reduced in Cirp-deficient inflammatory cells

(Fig. 4G) and more apoptosis was found in Cirp-/- immune cells than WT immune cells




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(Fig. 4H), which might at least partially contribute to the attenuated inflammatory

responses by Cirp-deficiency.

Cirp deficiency attenuated tumorigenesis in the murine CAC model

Chronic inflammation increases intestinal cancer risk in IBD (10). To investigate the

precise pathogenic mechanisms underlying IBD-associated colorectal carcinogenesis,

we used the AOM plus DSS mouse model to study the role of Cirp in CAC. In the

AOM/DSS protocol, a significant decrease was noted in the number and maximum size

of tumors in the Cirp-/- mice compared with WT mice (Fig. 5A and B). Histologic

examination of H&E-stained sections from the rolled-up colons revealed larger

adenomas with a complex tumor growth pattern in WT tumors compared with Cirp-/-

tumors (Fig. 5C). Extensive infiltration of inflammatory cells into the LP and

submucosal layer surrounding the tumors suggest the involvement of inflammatory

responses in the tumorigenesis seen in the AOM/DSS-treated mice (Fig. 5C). Colon

length was measured as one parameter to assess the severity of inflammation and was

found to be significantly longer in Cirp-/- mice than in WT mice (Fig. 5D). The

expression of TNF-α was decreased in non-tumorous tissue, but not in tumors, of Cirp-/-

mice challenged with AOM and DSS (Fig. 5E). Tumor and non-tumor cells would use

different mechanisms to regulate gene expression. In tumor cells, expression of TNF-α

might be upregulated in a Cirp-independent manner. IL-23 and IL-17 inhibits anti-tumor

immunity and promotes tumorigenesis (6-8). Expression of IL-23 and IL-17 was

decreased in Cirp-/- tumors and non-tumor colons compared to WT counterparts (Fig.

5E). Bcl-2 and Bcl-xL mRNA expression that is upregulated by Cirp in inflammatory

cells (Fig 4G) was significantly reduced in Cirp-deficient colons (Fig 5E).




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Cirp-deficiency decreased PCNA expression in DSS-treated colons whilst in established

tumors, neither apoptosis nor PCNA expression was significantly affected by Cirp

deletion (Fig. S6). The expression of stemness factor Sox2 was decreased in

Cirp-deficient colons and tumors compared with WT tissues (Fig. 5E and Fig. S5A).

Dclk1 is a candidate tumor stem cell marker in the gut (37). Deletion of Cirp decreased

the number of Dclk1+ cells at the tumor base (Fig. 5F).

Cirp expression was increased in the colonic mucosa of tumor-harboring mice given

DSS and AOM, whereas short-term inflammation induced by DSS administration for 7

days did not upregulate Cirp expression (Fig. 5G and Fig. S5B). Coupled with the

findings in humans (Fig 2B and 2E), these results suggest that Cirp is induced by

long-term intestinal inflammation.

Cirp promotes tumorigenesis through hematopoietic cell populations

To functionally characterize the contribution of different cell populations to colorectal

tumorigenesis, we created Cirp-chimeric mice using a combination of gamma

irradiation and BMT. Nontransplanted controls survived <2 weeks after irradiation,

indicating there was ablation of the endogenous marrow. Transplanted animals were

allowed to recover for 2 months prior to placing them on the AOM/DSS protocol. WT

mice rescued with Cirp-/- bone marrow had a significantly smaller tumor burden than

those rescued with WT bone marrow (Fig. 6A and 6B). Both WT and Cirp-/- mice

rescued with WT bone marrow had equivalent tumor sizes (Fig. 6B). Chimeras

harbouring Cirp-/- bone marrow diminished expression of TNF-α (Fig. 6C), which

indicates that Cirp in hematopoietic cells is involved in upregulation of TNF-α.

Cirp-chimeric mice with Cirp-deficient bone marrow showed a smaller number of




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Dclk1+ and Sox2+ cells in tumor than the WT mice (Fig. 6D and 6E). Taken together, at

least in this model, the absence of Cirp in hematopoietic cellular compartments protects

against AOM/DSS-induced tumorigenesis.

Discussion

The association between IBD and colorectal cancer is well established; the cumulative

risk of developing colorectal cancer after 20 years is 7% in UC and 8% in CD (10).

Optimal IBD management would reduce the risk of CAC (35). While it is clear that

chronic mucosal inflammation plays a causative role in the transition to adenocarcinoma,

the molecular link between inflammation and cancer remains to be elucidated. AOM is

a procarcinogen that is metabolically activated to a potent alkylating agent that forms

O6-methyl-guanine (38). Its oncogenic potential is markedly augmented in the setting of

chronic inflammation, such as that induced by repeated cycles of DSS treatment (39).

TNF-α, a key mediator of inflammation in IBD (3), contributes to tumorigenesis by

creating a tumor-supportive inflammatory microenvironment and through its direct

effect on malignant cells (40). Interactions between tumor and immune cells regulate

tumorigenesis. IL-23/IL-17 signaling has been correlated with promotion of tumor

growth, as well as IBD pathogenesis. In the tumor microenvironment, IL-23/IL-17

signaling suppresses anti-tumorigenic immune response during tumor initiation, growth,

and metastases (6-8). In the present study, Cirp deficiency decreased the production of

TNF-α and IL-23 in inflammatory cells and attenuated DSS-induced colitis. In the

murine CAC model, we found that TNF-α, IL-23 and IL-17 expression were increased

in Cirp-deficient mice and that Cirp was required for inflammatory-associated colonic




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carcinogenesis. Cirp expression was positively associated with the levels of TNF-α and

IL-23 in the colonic mucosa of IBD patients. Increased Cirp expression, seen in

refractory IBD, would promote tumorigenesis by enhancing TNF-α and IL-23

production. Given the contribution of Cirp in hematopoietic cells to tumor formation

(Fig. 6), Cirp likely promotes tumorigenesis through its action in inflammatory cells.

Adult somatic stem cells of the colon sustain self-renewal and are targets for cancer

initiation (41) and perturbation in stem cell dynamics is generally considered the first

step toward colon tumorigenesis. High levels of stemness factor Sox2 expression are

associated with poor prognosis and recurrence in patients with colorectal cancer (42). In

IBD patients, mucosal Cirp expression correlated with the expression of Sox2. We also

showed that Cirp is important for sustained expression of Sox2 in the colonic mucosa

during colorectal carcinogenesis. Cirp-deficiency decreased the number of cells positive

for an intestinal cancer stem cell marker Dclk1 at the tumor base. These data suggest a

possible function of Cirp in influencing stem cell behavior.

Cancer stem cells, the microenvironment and the immune system interact with each

other through cytokines. In the context of chronic inflammation, cytokines, secreted by

immune cells, activate the necessary pathways required by cancer stem cells (43). The

number of Sox2+ and Dclk1+ cells in tumor was decreased upon Cirp deletion in the

hematopoietic compartment (Fig. 6), suggesting that the absence of Cirp in

inflammatory cells decreased production/secretion of these cytokines. There were

statistically significant relationships between TNF-α and Dclk1 expression and between

IL-23/IL-17 and Sox2 expression in colonic mucosa of UC patients (data not shown).

Thus, Cirp-driven immune responses such as activation of TNF-α and IL-23/IL-17

signaling would affect proliferation of stem cells and increase the expression of stem




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cell markers. It should be noted, however, that the direct causal link between Cirp and

the stem cell markers has not been established in this study. In this regards, the reduced

expression of the stem cell markers such as Dclk1 and Sox2 seen in the absence of Cirp

might be due to the secondary effects associated with reduced inflammation.

Apoptotic cell death has been implicated as a major homeostatic and pathogenic

mechanism of the intestinal epithelium (2). The lower susceptibility to apoptosis

observed in the Cirp-/- intestinal epithelial mucosa in our in vivo experiment was

unexpected because a previous report showed that Cirp attenuates TNFα-mediated

apoptosis by activating ERK and NF-κB in murine embryonic fibroblasts (27). However,

expression of Cirp did not affect the sensitivity of murine embryonic fibroblasts to

busulfan, and the numbers of apoptotic testicular cells were not different between Cirp-/-

and WT mice after busulfan treatment (28). Thus, the role of Cirp may vary depending

on cell type and kind of stimuli. In fact, in the DSS-treated colon, Cirp-deficiency did

not attenuate ERK activity (Fig. S4A). In Cirp-/- mice, more inflammatory cells died due

to decreased Bcl-2 and Bcl-xL expression than in WT mice (Fig. 4G and 4H), which

would attenuate inflammatory response in Cirp-/- mice. Cell death and inflammation are

intimately linked through a self-amplifying loop, making it difficult to distinguish

between causes and effects. The attenuated mucosal immune activity due to augmented

apoptosis of inflammatory cells likely contributed to the decreased apoptosis of

epithelial cells in Cirp-deficient colon.

Bcl-2-mediated apoptosis resistance in inflammatory cells has been shown to

attenuate therapeutic efficacy and exacerbate inflammation in IBD (33, 34). In

chronically inflamed mucosa seen in refractory UC, Cirp expression is induced in

inflammatory cells, which likely inhibits the apoptosis of inflammatory cells, augments




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pro-inflammatory cytokine production and treatment resistance via the upregulation of

Bcl-2 and Bcl-xL expression. Thus, persistent inflammation resulting from insufficient

treatment might further drive resistance to therapy through increased expression of Cirp

and subsequent attenuated apoptosis in inflammatory cells. Hypoxia that is enhanced in

chronic inflammatory diseases including IBD upregulates Cirp expression by a

mechanism that involves neither HIF-1 nor mitochondria (20). This may be one

explanation for Cirp induction by chronic inflammation. However, the exact

mechanisms by which long-term inflammation upregulates Cirp expression remain to be

elucidated.

It has been reported that Cirp released into the circulation stimulates the release of

TNF-α from macrophages via TLR4 and NF-κB activation, and triggers an

inflammatory response to hemorrhagic shock and sepsis (25). Here we have shown that

in bone marrow-derived macrophages, the presence of Cirp increased IκBα

phosphorylation. NF-κB activation would be one of mechanisms by which Cirp

produces proinflammatory cytokines such as TNF-α, IL-17 and IL-23 and upregulates

expression of anti-apoptotic genes such as Bcl-2 and Bcl-xL (Fig. 4 and 5). A recent

study reported the involvement of Cirp in regulating expression of IL-1β, another

NF-κB target gene, in cultured fibroblasts (44). In bone marrow-derived macrophages,

IL-1β mRNA level was decreased in the absence of Cirp (data not shown). Although in

DSS-induced colitis, Cirp protein was not detected in the blood (data not shown), it is

conceivable that Cirp released from injured epithelial cells could function as

damage-associated molecular pattern molecules in situ to activate NF-κB in immune

cells of the colon. Furthermore, Cirp can bind the 3'untranslated region of specific

transcripts to stabilize them and facilitate their transport to ribosomes for translation




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(22-24). Cirp might regulate expression of cytokines and anti-apoptotic genes

posttranscriptionally as well.

Given the long-term impact of the natural history and treatment of IBD, cancer risk

is a major lifelong concern for patients and gastroenterologists. Early detection of

colitis-associated cancer/dysplasia is typically achieved by colonoscopic surveillance

with multiple biopsies and alternatively by chromoendoscopy with targeted biopsies of

all suspect areas. It has been reported that in patients with extensive colitis, surveillance

should start after colonoscopy screening (8-10 years after disease onset) and be

performed every 2 years for 20 years, then once or twice a year for the next 10 years of

disease duration (45). However, such surveillance programs have a number of

limitations such as low yield, high cost, invasiveness, incomplete patient enrollment,

sampling variations, and poor agreement in histopathological interpretation (46). If we

can reliably predict an individual’s risk of CAC so that surveillance strategies can be

appropriately personalized, surveillance programs would make much progress. A

number of molecular markers for predicting CAC have been reported (47-49) but are

not feasible yet for practical management of IBD patients. Here we showed significantly

increased Cirp expression in mucosal specimens from patients with refractory IBD that

is reported to be associated with increased cancer risk (9). Furthermore, in the murine

CAC model, longstanding colonic inflammation increased Cirp expression, which led to

enhanced AOM/DSS-induced colorectal tumorigenesis. Cirp expression reflects the

presence of refractory inflammation and is therefore a potential marker for predicting

the risk of CAC development. Analyzing the Cirp level in colonoscopy specimens may

increase the positive identification rate of IBD patients with a high risk for developing

CAC. A future large-scale study of Cirp in IBD patients with different duration and




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anatomical extent of the disease will be crucial for determining whether Cirp status can

be utilized to predict the risk of cancer and prognosis of IBD patients.

Taken together, Cirp, whose expression is upregulated by chronic inflammation in

humans and mice, enhances the inflammatory response and tumorigenesis by increasing

Bcl-2 and Bcl-xL expression and TNF-α and IL-23/IL-17 production in inflammatory

cells. Suppression and measurement of Cirp expression is a promising approach for

advanced treatment and personalized management of IBD patients.

Acknowledgements

This research was supported by grants from the Takeda Science Foundation, the Japan

Foundation for Research and Promotion of Endoscopy, and the Smoking Research

Foundation of Japan, as well as a Grant-in-Aid for Scientific Research (26460979) and

Health Labour Sciences Research Grant.




21

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27

Figure legends

Figure 1. Association between Cirp and the expression of Bcl-2 and various stem cell

markers in the colonic mucosa of patients with ulcerative colitis (UC). Scatter plot of

relative mRNA levels of Cirp and the respective genes in human colonic mucosa.

Figure 2. Cirp expression is increased in the colonic mucosa of patients with refractory

ulcerative colitis (UC). (A) Endoscopic images of refractory and non-refractory active

UC specimens with a magnified inset. (B-D) Expression of Cirp (B), Sox2 (C), and

TNF-α (D) mRNA in normal colonic mucosa (control, n = 30), colonic mucosa of

patients with non-refractory active UC (non-refractory, n = 98), and those with

refractory UC (refractory, n = 67), as determined by quantitative real-time qPCR. P

values were calculated by post-hoc Tukey-Kramer HSD multiple comparison. The F and

p values for the ANOVO test are as follows: F (2, 192) = 11.98; p<0.0001 (B), F (2,

192) = 10.29; p<0.0001 (C) and F (2, 192) = 13.70; p<0.0001 (D). (E) Representative

images of immunohistochemical findings in human colonic mucosa of patients without

UC and those with non-refractory and refractory UC. Scale bar, 50 μm.

Figure 3. Susceptibility to inflammation is decreased in Cirp-/- mice challenged with

DSS. (A) Representative photographs of H&E-stained and PAS-stained colons of WT

and Cirp-/- mice 7 days after the initiation of DSS administration (original magnification,

×200). Scale bar, 50 μm. (B) Inflammatory cell infiltration into colonic tissues of WT

and Cirp-/- mice 7 days after the initiation of DSS administration. Scoring was

performed as described in the Materials and Methods. n=6 per group. (C)

Representative images of immunohistochemical detection of F4/80, a marker for




28

macrophages, in colonic tissue. Scale bar, 100 μm. (D) Histologic scoring of epithelial

injury in colons. n=6 per group. (E) TUNEL staining of colonic tissues from

DSS-treated mice (original magnification, ×200). (F) The apoptotic index was measured

by counting TUNEL signals in 100 randomly selected crypts. Results are expressed as

means + SEM (n = 4 per group). *P < 0.05 compared with WT mice.

Figure 4. Cirp contributes to TNF-α and IL-23 production and Bcl-2 and Bcl-xL

exprsssion in inflammatory cells. (A-C) Relative levels of TNF-α (A), IL-1β (B) and

IL-23 (C) in colonic tissues from mice treated with DSS, as determined by real-time

qPCR (n = 8 per group). Expression level in colonic tissues from non-treated WT mice

was set as 1. (D, E) Effect of Cirp on gene expression in colonic lamina propria (LP)

cells (D) and macrophages (E). LP cells and macrophages were isolated and cytokine

mRNA expression was analyzed by real-time qPCR. The mRNA expression levels in LP

cells or macrophages from non-treated WT mice were set as 1, respectively. Results are

expressed as means + SEM (n = 4 per group). *P < 0.05 compared with WT mice. (F)

Inflammatory cell infiltration into colonic tissues of WT and Cirp-/- mice 7 days after

the initiation of DSS administration with or without anti-TNF-α antibody treatment.

Scoring was performed as described in the Materials and Methods. n=3 per group. *P <

0.05 compared with control. (G) Effect of Cirp on gene expression in colonic LP cells.

LP cells were isolated and cytokine mRNA expression was analyzed by real-time qPCR.

The mRNA expression levels in LP cells from non-treated WT mice were set as 1. (H)

Representative images of immunohistochemical detection of E-cadherin, a marker for

epithelial cells, and TUNEL staining of colonic tissues from DSS-treated mice.




29

Figure 5. Cirp deficiency affects colonic tumorigenesis in a murine model of CAC. (A)

Typical examples of macroscopic tumorigenesis in the CAC model. Colons were cut

longitudinally. (B) Tumor number and maximum size (WT mice, n = 8; Cirp-/- mice, n =

8). (C) Typical examples of microscopic tumorigenesis in the CAC model with

magnified insets. Scale bar, 500 μm. (D) Colon length after treatment with AOM and

DSS. (E) RNA was extracted from non-tumorous colon and tumor tissues. Relative

amounts of mRNA, as determined by real-time qPCR normalized to the amount of actin

mRNA. The amount of each mRNA in the untreated colon was given an arbitrary value

of 1.0. (F) Immunohistochemistry findings of colon sections of AOM/DSS-treated WT

and Cirp-/- mice. The tumor bases stained with anti-Dclk1 antibody were identified by

confocal microscopy. Scale bar, 15 μm. (G) RNA was extracted from the colonic tissues

of WT mice 7 days after the initiation of DSS administration (DSS, n = 8) and

non-tumorous colon and tumor tissues of WT mice after administration of AOM and

DSS (DSS+AOM, n = 6). Relative amounts of mRNA, as determined by real-time

qPCR and normalized to the amount of actin mRNA. The mean value of mRNA in

untreated colon (control, n = 5) was given an arbitrary value of 1.0. P values were

calculated by post-hoc Tukey-Kramer HSD multiple comparison.

Figure 6. Cirp promotes tumorigenesis through hematopoietic cell populations. WT

mice with WT or Cirp–/– bone marrow (BM) and Cirp–/– mice with WT or Cirp–/– BM

were generated by bone marrow transplantation. Number (A) and maximum size (B) of

the tumors (WT-BM/WT mice, n = 8; Cirp-/--BM/WT mice, n = 7; WT-BM/Cirp-/- mice,

n = 7; Cirp-/--BM/ Cirp-/- mice, n = 6). P values were calculated by post-hoc

Tukey-Kramer HSD multiple comparison. (C) RNA was extracted from non-tumorous




30

colon. Relative amounts of mRNA, as determined by real-time qPCR normalized to the

amount of actin mRNA. The amount of TNF-α mRNA in the untreated colon was given

an arbitrary value of 1.0. n=6 per group. (D) Representative images of

immunohistochemical detection of Dclk1 at the tumor base and Sox2 in tumors. Scale

bar, 100 μm. (E) The number of Dclk1+ cells at the tumor base and Sox2+ in tumors.




Published OnlineFirst September 3, 2014.Cancer Res Toshiharu Sakurai, Hiroshi Kashida, Tomohiro Watanabe, et al. tumorigenesis in colitis-associated cancerStress response protein Cirp links inflammation and

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