Prevention of Colitis and Colitis-Associated ... · the association between chronic inﬂammation (colitis) and colorectal cancer (4, 6). In addition, both sporadic and herita-ble

Cancer Therapy: Preclinical

Prevention of Colitis and Colitis-AssociatedColorectal CancerbyaNovelPolypharmacologicalHistone Deacetylase InhibitorTzu-Tang Wei1, Yi-Ting Lin1, Ruo-Yu Tseng1, Chia-Tung Shun2,3, Yu-Chin Lin1,4,5,Ming-Shiang Wu6, Jim-Min Fang7, and Ching-Chow Chen1

Abstract

Purpose: Colorectal cancer is a worldwide cancer with risingannual incidence. Inflammation is a well-known cause of colo-rectal cancer carcinogenesis. Metabolic inflammation (metaflam-mation) and altered gut microbiota (dysbiosis) have contributedto colorectal cancer. Chemoprevention is an important strategy toreduce cancer-related mortality. Recently, various polypharma-cologic molecules that dually inhibit histone deacetylases(HDAC) and other therapeutic targets have been developed.

Experimental Design: Prevention for colitis was examined bydextran sodium sulfate (DSS) mouse models. Prevention for colo-rectal cancer was examined by azoxymethane/dextran sodiumsulfate (AOM/DSS) mouse models. Immunohistochemical stain-ing was utilized to analyze the infiltration of macrophages andneutrophils andCOX-II expression inmouse tissue specimens. Theendotoxin activity was evaluated by Endotoxin Activity Assay Kit.

Results: We synthesized a statin hydroxamate that simulta-neously inhibited HDAC and 3-hydroxy-3-methylglutaryl coen-

zyme A reductase (HMGR). Its preventive effect on colitis andcolitis-associated colorectal cancer in mouse models was exam-ined. Oral administration of this statin hydroxamate could pre-vent acute inflammation in the DSS-induced colitis and AOM/DSS–induced colorectal cancer with superior activity than thecombination of lovastatin and SAHA. It also reduced proinflam-matory cytokines, chemokines, expression of COX-II, and cyclinD1 in inflammation and tumor tissues, as well as decreasing theinfiltration of macrophages and neutrophils in tumor-surround-ing regions. Stemness of colorectal cancer and the release ofendotoxin in AOM/DSS mouse models were also attenuated bythis small molecule.

Conclusions: This study demonstrates that the polypharma-cological HDAC inhibitor has promising effect on the chemopre-vention of colorectal cancer, and serum endotoxin level mightserve as a potential biomarker for its chemoprevention. Clin CancerRes; 22(16); 4158–69. �2016 AACR.

IntroductionColorectal cancer is a worldwide cancer, causing significant

morbidity and mortality, and chemoprevention is one of thepotentials to reduce cancer-related mortality by suppressing theinitial phase of carcinogenesis or the progression of premalignantcells (1). Meta-analysis studies have shown that aspirin couldreduce the incidence of colon polyps and cancer (2). Themechan-isms were in part through inhibition of COX-II–related pathways

and normalizing EGFR expression (3). However, adverse effectsprohibit its clinical use. Thus, new strategy for chemopreventionof colorectal cancer meets an unmet medical need.

Inflammation through inflammation–dysplasia–carcinomaprocess is an important cause of carcinogenesis to inducecolorectal cancer (4, 5). Inflammatory bowel disease (IBD)referring to both Crohn's disease and ulcerative colitis is achronic disorder. Patients with IBD exhibiting a 2- to 8-foldhigher risk of colorectal cancer, is a classic example to highlightthe association between chronic inflammation (colitis) andcolorectal cancer (4, 6). In addition, both sporadic and herita-ble colorectal cancer are also related to inflammation (6, 7).Metabolic syndrome (i.e., hypertension, type II diabetes, hyper-lipidemia, and obesity) commonly resulted from excess nutri-ents and energy has been found to induce a low-grade, chronic,metabolically linked inflammatory state termed metaflamma-tion (8). Patients having metabolic syndromes show higherincidence of colorectal cancer and higher recurrence rate ofcolon adenoma that demonstrated the close associationbetween inflammation and colorectal cancer (8). Epidemiolo-gic studies reported that anti-inflammatory medications effec-tively reduce the incidence of colorectal cancer among IBDpatients (9, 10). Therefore, inhibition of inflammation is apotential strategy to prevent cancer initiation.

Huge amount of bacteria flora inhabited in the intestine inter-acting with mucosa maintaining gut immunity and homeostasis

1Department of Pharmacology, National Taiwan University College ofMedicine, Taipei, Taiwan. 2Graduate Institute of Forensic Medicine,National Taiwan University College of Medicine, Taipei, Taiwan.3DepartmentofPathology,National TaiwanUniversityHospital,Taipei,Taiwan. 4Department of Oncology, National Taiwan University Hospi-tal, Taipei, Taiwan. 5Department of Internal Medicine, Far-EasternMemorial Hospital, New Taipei City, Taiwan. 6Division of Gastroenter-ology, Department of Internal Medicine, National Taiwan UniversityHospital, Taipei, Taiwan. 7Department of Chemistry, National TaiwanUniversity, Taipei, Taiwan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor:Ching-ChowChen, National Taiwan University College ofMedicine, No. 1, Jen-Ai Road, 1st Section, Taipei 10051, Taiwan. Phone: 8862-2312-3456, ext. 88321; Fax: 886-2-23947833; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2379

�2016 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 22(16) August 15, 20164158

on August 8, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from on August 8, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from on August 8, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/



(11). Recent studies suggest that imbalance of bacterial flora,termed dysbiosis, favors higher abundance of gram-negativebacteria (GNB; refs. 4, 12, 13). Endotoxin, which is a lipopoly-saccharide (LPS) from the cell wall of GNB, is associated withvarious chronic diseases, such as atherosclerosis, diabetes, andcancers (13). The release of endotoxin by GNB is an importantmechanism of dysbiosis-induced colorectal cancer (14). In addi-tion to endotoxin, cancer stem cell (CSC) is another crucial factorfor colorectal cancer initiation (15). Chronic gut inflammationleads to crypt damage and repeated regeneration, resulting in CSCexpansion leading to colorectal tumorigenesis (16, 17).

Statins are 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase (HMGR) inhibitors treating hypercholesterol-emia in patients with coronary artery and atherosclerotic diseases(18, 19). Theyhave also shownpromise in cancer prevention fromobservational and preclinical and certain aspects of randomizedcontrolled studies (20). We first demonstrated statins with acarboxylic acid–containing long chain possessing the structuresimilar to hydroxamate that also inhibits histone deacetylase(HDAC) activity, which is an attractive target against cancer(21, 22). To further improve statins' activity against HDAC, threestatin hydroxamates were designed to inhibit class I and II HDACsand HMGR (23). We named compound 12, 13, and 14 asJMF3086, 3171, and 3173, respectively, in this study. Recently,the strategy of developing polypharmacological molecules thatdually inhibit HDACs and other therapeutic targets has beenreported (24).

Oral administration of JMF3086 attenuated dextran sodiumsulfate (DSS)–induced colitis and azoxymethane (AOM)/DSS–induced colorectal cancer in mice. A positive correlation betweenHMGR and HDAC activity and prevention of colorectal cancerwas seen. JMF3086 exerted anti-inflammatory effect and inhibitedthe in vivo release of endotoxin. Furthermore, the stemness ofcolorectal cancer was inhibited in vivo. These results show that ourpolypharmacological molecule JMF3086 targeting HDACs andHMGR possesses great potential for the prevention of colitis andcolitis-associated colorectal cancer.

Materials and MethodsReagents

Lovastatin was obtained from Lotus Pharmaceutical Co. SAHAwas obtained and purchased from Merck. Anti-acetyl-histone H3and anti-acetyl-histone H4 antibodies were obtained from Milli-pore. Anti-acetyl-tubulin antibody was obtained from Sigma.Anti-PARP1/2 antibody was purchased from Santa Cruz Biotech-nology. Anti-caspase-3 antibody and anti-BAX antibody werepurchased fromCell Signaling Technology. Anti-b-actin antibodywas purchased from GeneTex. HDAC Fluorometric Assay/DrugDiscovery Kit (AK-500) was purchased from Biomol. HMG-CoAreductase activity kit (CS-1090) and AOM were purchased fromSigma. DSS was purchased from MP Biomedicals. EndotoxinActivity Assay Kit was purchased from Spectral Diagnostics.

MiceC57BL/6, BALB/c, and NOD/SCID mice were obtained from

the National Laboratory Animal Center (Taipei City, Taiwan).Mice were performed in accordance with protocols approved bythe Institutional Animal Care and Use Committee of the Collegeof Medicine, National Taiwan University (Taipei, Taiwan).

Animal models of DSS-induced acute colitisThe DSS-induced colitis animal model exhibits many pheno-

types that are relevant tohumanulcerative colitis, including inflam-mation and ulceration of the colonic mucosa (4). Colitis wasinduced in seven-week-old male C57BL/6 mice by adding DSS totheir drinking water for 5 days, followed by switching to regulardrinking water. In the preventive model, test compounds weredissolved in corn oil and administered orally 7 days before, during,and after 3.5% (w/v) DSS treatment (Fig. 1A). In the therapeuticmodel, test compoundsweredissolved incornoil andadministeredorally 5 days following the 2.5% (w/v) DSS treatment (Fig. 2A).

Animal model of AOM/DSS–induced colorectal cancerAOM in conjunction with the additional inflammatory stim-

ulus of DSS results in tumor development that is restricted to thecolon in mice (4, 6). This model is widely utilized to recapitulatehuman colorectal cancer because it results in inflammation andulceration of the entire colon, similar to what is observed inpatients (6). Colorectal cancerwas induced inmale C57BL/6micevia the intraperitoneal injection of AOM (12.5 mg/kg) while themice weremaintained with a regular diet and drinking water for 7days and then subjecting the mice to 3 cycles of DSS treatment,with each cycle consisting of the administration of 3.5% DSS for5 days, followed by a 14-day recovery period with regular water(Fig. 3A). In the preventive model of AOM/DSS–induced colo-rectal cancer, test compounds were dissolved in corn oil andadministered orally 5 days a week for 8 weeks, simultaneouslywith AOM exposure.

Clinical assessment of DSS-induced acute colitis and AOM/DSS–induced colorectal cancer

Body weight, the presence of occult or gross blood in therectum, and stool consistency were determined daily in the mice.The weight change during the experiment was calculated as thepercent change in weight compared with the baseline measure-ment. Bleeding was scored as 0, when there was no blood in theHemoccult test; 1, for a positive Hemoccult result; 2, for slightbleeding; or 3, for gross bleeding. Regarding stool consistency,

Translational Relevance

Colorectal cancer is a worldwide cancer, causing significantmorbidity and mortality, and chemoprevention is one of thepotentials to reduce cancer-relatedmortality.Here,we design apolypharmacologic small molecule, statin hydroxamate, thatdually inhibits histonedeacetylases (HDAC)and3-hydroxy-3-methylglutaryl coenzyme A reductase. It attenuated dextransodium sulfate (DSS)–induced colitis and azoxymethane/DSS(AOM/DSS)–induced colorectal cancer inmice through reduc-ing proinflammatory cytokines, chemokines, expression ofCOX-II, and cyclin D1 in inflammation and tumor tissues, aswell as decreasing the infiltration of macrophages and neu-trophils in tumor-surrounding regions. Stemness ofColorectalcancer and the release of endotoxin in AOM/DSS mousemodels were also attenuated by this small molecule. Theseresults demonstrate that the polypharmacologic HDAC inhib-itor has promising effect on the chemoprevention of Colorec-tal cancer, and serumendotoxin levelmight serve as a potentialbiomarker for its chemoprevention.

Colorectal Cancer Chemoprevention by Statin Hydroxamate

www.aacrjournals.org Clin Cancer Res; 22(16) August 15, 2016 4159

on August 8, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from


A

D

F

0

20

40

60

80

0

1,000

2,000

3,000

0102030405060

0

100

200

300

400

** **

TNFa##

** ***

IFNg

##

IL12

*** **

##*

IL6

*** *

##

**

*

B

Cha

nge

in w

eigh

t (%

)B

leed

ing

scor

e (0

–3)

Dia

rrhe

a sc

ore

(0–3

)

***

Days

Days

Days

3.5% DSS

C

E

pg/m

L

Vehicle (oil), n = 15

Vehicle, n = 15

JMF3086 50 mg/kg, n = 10

JMF3086 50 mg/kg, n = 10

SAHA 50 mg/kg, n = 9

SAHA 50 mg/kg, n = 9

LOVA 50 mg/kg, n = 7LOVA+SAHA, n = 8

LOVA + SAHA, n = 8

LOVA 50 mg/kg, n = 7

D0 D2 D4 D6 D8 D10 D12 D14 D16

H2O H2ODSS

Drug

3.5% DSS

3.5% DSS

3.5% DSS

Sacrifice

Co

ntr

ol

LO

VA

SA

HA

+ S

AH

AL

OVA

Veh

icle

JMF

308

650

mg

/kg

50 m

g/k

g

50 m

g/k

g

Control

Control

LOVA

LOVA

SAHA

SAHA

+ SAHA

+ SAHA

LOVA

LOVA

Vehicle

Vehicle

JMF 3086

JMF 3086

50 mg/kg

50 mg/kg

50 mg/kg

50 mg/kg

50 mg/kg

50 mg/kg

0 2 4 6 8 10 12 14 165

0

–5

–10

–15

–20

–25

3.5% DSS

3.5% DSS

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

3.53.02.52.01.51.00.50.0

0 2 4 6 8 10 12 14 16

0 2 4 6 8 10 12 14 16

Control, n = 7

9

8

7

6

5

Col

on le

ngth

(cm

)

H&

EC

OX-

IILy

-6G

F4/8

0

Figure 1.

JMF3086 prevents acute colitis in vivo. A, schematic overview of the experimental design. B, changes in body weights (top), clinical bleeding scores (middle),and clinical diarrhea scores (bottom) are shown. � , P < 0.05; �� , P < 0.01 versus vehicle. C, representative whole colons (top) and colon length (bottom)are shown. Control mice received distilled water only. Scale bar, 2 cm. ##, P < 0.01 versus control; �� , P < 0.01 versus vehicle. D, colon sections were counterstainedwith H&E, and high-magnification images of the black-boxed area are presented in the bottom row. Scale bar, 250 mm. E, colon sections were immunostainedwith anti-F4/80, anti-Ly-6G, and anti-COX-II antibodies. Scale bar, 250 mm. F, the level of various cytokines in colon culture supernatants was seen. ##, P < 0.01 versuscontrol; � , P < 0.05 and �� , P < 0.01 versus vehicle.

Wei et al.

Clin Cancer Res; 22(16) August 15, 2016 Clinical Cancer Research4160



A

F

B

C

Vehicle, n = 5JMF3086 50 mg/kg, n = 5

Control, n = 2

LOVA 50 mg/kg, n = 5SAHA 50 mg/kg, n = 5LOVA+SAHA, n = 5

##GM-CSF

pg/m

L

pg/m

L

pg/m

L* ** ##

* *

##

**

Dia

rrhe

a sc

ore

(0–3

)

Days

***

0.0

0.5

1.0

1.5

2.0

2.5

3.0

1086420

2.5% DSS Drug

**

Days

***

Ble

edin

g sc

ore

(0–3

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

1086420

2.5% DSS Drug

Cha

nge

in w

eigh

t (%

)

Days

–14–12–10

–8–6–4–2

0246

1086420

Vehicle (n = 5)JMF3086 (n = 5)SAHA (n = 5)Lovastatin (n = 5)Lova + SAHA (n = 5)

*

2.5% DSS Drug

5

6

7

8

9

10

Col

on le

ngth

(cm

)

H&E

H2ODSS

D0 D2 D4 D6 D8 D10

Drug Sacrifice

2.5% DSS

2.5% DSS

2.5% DSS

Co

ntr

ol

LO

VA

SA

HA

+ S

AH

AL

OVA

Veh

icle

JMF

308

650

mg

/kg

50 m

g/k

g

50 m

g/k

g

ControlLOVA SAHA

+ SAHALOVA

VehicleJMF 308650 mg/kg 50 mg/kg 50 mg/kg

Control

LOVA SAHA

+ SAHA

LOVA

Vehicle

JMF 3086

50 mg/kg 50 mg/kg 50 mg/kg

Ly-6

G

TNFa IL1b8070605040302010

0

400350300250200150100

500

1,000900800700600500400300200100

0

D

E

Figure 2.

JMF3086 treats acute colitis in vivo. A, schematic overview of the experimental design. B, changes in body weights (top), clinical bleeding scores (middle), and clinicaldiarrhea scores (bottom) are shown. � , P < 0.05; ��, P < 0.01; and �� , P < 0.001 versus vehicle. C, representative whole colons (top) and colon length (bottom) areshown. Controlmice received distilledwater only. Scale bar, 2 cm.D, colon sectionswere counterstainedwith H&E, and high-magnification images of the black-boxed areaare presented in the bottomrow. Scale bar, 100mm.E, colon sectionswere immunostainedwith anti-Ly-6G antibodies. Scale bar, 100mm.F, the level of various cytokines incolon culture supernatants was seen. ##, P < 0.01 versus control; �, P < 0.05 versus vehicle. GM-CSF, granulocyte macrophage colony-stimulating factor.





0 points were given for well-formed pellets, 1 point for semi-formed stools that did not adhere to the anus, 2 points for pastystools, and 3 points for liquid stools that adhered to the anus.

HDAC activity assayThe HDAC activity was performed using HDAC Fluorescent

Activity Assay Kit. Total lysate frommouse tissue was mixed withFluor-de-Lys substrate for 10minutes at 37�C, followed by addingdeveloper to stop the reaction. Fluorescence was measured byfluorometric readerwith excitation at 360nmand emission at 460nm. The IC50 values were calculated from SigmaPlot software.

HMG-CoA reductase activity assayThe HMG-CoA reductase activity was performed using HMG-

CoA Reductase Assay Kit. Total lysate from mouse tissue wasmixed with NADPH and HMG-CoA and incubated for 5 minutes

at 37�C. The absorbance at 340 nmwasmeasured. The IC50 valueswere calculated from SigmaPlot software.

IHC assayIHC was performed with One Step Polymer-HRP Detection Kit

(BioGenex) on sections from 10% paraffin-embedded samplesaccording to the manufacturer's protocols. Pictures were acquiredusing TissueFAXS (TissueGnostics), and the results of DAB-pos-itive cells were presented as scattergrams plot using HistoQuestsoftware (TissueGnostics). The cut-off values for backgroundstaining were chosen manually using the forward/backwardgating tools of the software.

Endotoxin activity assayThe endotoxin activity was performed using Endotoxin Activity

Assay Kit. Serum from mouse whole blood was mixed with

A

C

Days

Ble

edin

g sc

ore

(0–3

)C

hang

es o

f b

ody

wei

ght (

%)

0.00.51.01.52.02.53.03.5

5550454035302520151050

AOM+JMF

DSS DSS DSS

*****

Dia

rrhe

a sc

ore

(0–3

)

Days

0.00.51.01.52.02.53.03.5

5550454035302520151050

AOM+JMF

DSS DSS DSS

******

BVehicle, n = 19


JMF3086 25 mg/kg, n = 10JMF3086 50 mg/kg, n = 20

SAHA 50 mg/kg, n = 6LOVA + SAHA, n = 6

H2O H2O H2ODSS DSS DSS

W0 W1 W2 W3 W4 W5 W6 W7 W8

SacrificeAOM and JMF3086

AOM+DSS (week 8)

Control LOVA SAHA+SAHALOVAVehicle JMF 3086 JMF 3086

25 mg/kg 50 mg/kg50 mg/kg 50 mg/kg

1110

98765C

olon

leng

th (c

m)

Co

ntr

ol

LO

VA

SA

HA

+ S

AH

AL

OVA

Veh

icle

JMF

308

6

JMF

308

6

25 m

g/k

g

50 m

g/k

g

50 m

g/k

g

50 m

g/k

g

AOM+JMF

DSSDSS

DSS

Days

20

10

0

–10

–20

0 5 10 15 20 25 30 35 40 45 50 55

Figure 3.

Preventive effect of JMF3086 on the colitis symptoms in AOM/DSS mouse models. A, experimental timeline for AOM/DSS–induced colorectal cancer mousemodel. B, changes in body weights (top), clinical bleeding scores (middle), and clinical diarrhea scores (bottom) are shown. Error bars, SE. � , P < 0.05;�� , P < 0.01 versus vehicle; two-sided student t test. C, representative whole colons (top) and colon length (bottom) are shown. Scale bar, 2 cm. Error bars,SE. � , P < 0.05; ��, P < 0.01 versus vehicle; ##, P < 0.01 versus control; two-sided student t test.

Wei et al.




control standard endotoxin and limulus amoebocyte lysatereagent water at 37�C. The absorbance at 405 nm was measured.

Statistical analysisThe SPSS program (SPSS Inc.) was used for all statistical

analyses. Statistical analysis was performed by two-sided Studentt test. Data shown were representative of at least three indepen-dent experiments. Quantitative data are presented asmeans� SD.

Additional methodsDetailed methodology is described in the Supplementary

Material.

ResultsStatin hydroxamates prevent inflammation in the DSS-inducedcolitis

As inhibition of inflammation is a potential strategy to preventcancer initiation, the acute colitis induced by DSS in C57BL/6mouse model was employed to evaluate the anti-inflammationefficacy of JMF3086. JMF3086 (50 mg/kg) was orally adminis-tered for 7 days before treatment with 3.5% DSS began and wascontinued until the end of the experiment (Fig. 1A). It preventedthe colitis symptoms (i.e., body weight loss, diarrhea, and rectalbleeding) and was more effective than the combination oflovastatin and SAHA (Fig. 1B).

The reduction in colon length that occurred inDSS-treatedmicewas prevented by JMF3086 (Fig. 1C). Histopathologically, DSS-treated mice exhibited complete destruction of the epithelialarchitecture with a loss of crypts and epithelial integrity andsignificant infiltration of neutrophils (Ly-6G) but not of macro-phages (F4/80). In JMF3086-treated mice, the crypt architecturewas maintained, and only a mild infiltration of neutrophils inthe mucosa was detected (Fig. 1D and E). Furthermore, elevatedCOX-II expression was observed in the epithelial cells of DSS-treated mice, and this increase was prevented by JMF3086(Fig. 1E).DSS-induced acute colitis is predominantly characterizedby a cytokine response (25). TNFa, IFNg , IL6, and IL12 secretionfrom colons in the DSS-treated mice were found to be markedlyincreased and prevented by JMF3086 treatment (Fig. 1F).

The preventive effect of JMF3171 and JMF3173 on DSS-induced colitis was also examined and found to exert nice efficacyin colitis symptoms, as well as the reductions of colon length,infiltration of neutrophils, destruction of the epithelial architec-ture, and cytokine releases (Supplementary Fig. S1).

JMF3086 treats DSS-induced colitisWe further examined the efficacy of JMF3086 in treating DSS-

induced colitis in mouse models. The colitis symptoms wereinhibited by JMF3086, which was more effective than the com-bination of lovastatin and SAHA (Fig. 2B andC).Microscopically,JMF3086 reduced DSS-induced colonic ulceration (Fig. 2D).Furthermore, infiltration of neutrophils as well as the secretionof proinflammatory cytokines and chemokines in the colonswas attenuated by JMF3086 (Fig. 2E and F). Elevated serumendotoxin level found in DSS mice was reduced by JMF3086(Supplementary Fig. S2).

JMF3086 attenuates the colitis symptoms and tumor growth inAOM/DSS–induced colorectal cancer mouse models

As JMF3086 could prevent and treat acute inflammation in theDSS-induced colitis in mice, the chemoprevention of JMF3086

was further evaluated in AOM/DSS–induced colorectal cancermouse models. JMF3086 (25 or 50 mg/kg) was administeredorally 5 days per week concurrent with AOM injections (Fig. 3A).The effect of lovastatin (50 mg/kg) and SAHA (50 mg/kg) eitherindividually or in combination was compared. Symptomaticparameters, such as loss of body weight, diarrhea, and rectalbleeding, were observed in mice after AOM/DSS treatment, andJMF3086 prevented the occurrence of these symptoms (Fig. 3B).Colon lengthwas decreased inAOM/DSS–treatedmice, but not inthose treated with JMF3086 (Fig. 3C).

Polyps and colon tumors were macroscopically observed andcounted, and H&E staining revealed adenocarcinomas with dys-plasia in AOM/DSS–treated mice (Fig. 4A and B and Supplemen-tary Fig. S3A). JMF3086 at a dose of 50 mg/kg decreased tumornumbers and sizes and protected against AOM/DSS–induceddysplasia/adenocarcinoma lesion to surface tumor necrosis(Fig. 4A and B).

To examine whether HMGR and HDAC were inhibited byJMF3086 in vivo, their activity was analyzed in cell lysates fromcolon tissue and found to be increased in AOM/DSS–inducedcolorectal cancer. JMF3086 inhibited these effects and inducedacetylations of histone H3, H4, and tubulin. Its accumulation incolon tissues detected by LC/MS-MS and ESI-MS analyses wasseen (Fig. 4C and Supplementary Figs. S3B and S4A–S4C). Therewas a positive correlation betweenHMGR andHDAC activity andtumor number and size (Fig. 4D), indicating that inhibition ofboth activities contributed to the prevention of JMF3086 onAOM/DSS–induced colorectal cancer in mice.

JMF3086 reduces the mRNA expression of proinflammatorycytokines/chemokines and intracolonic infiltration ofmacrophages andneutrophils in AOM/DSS–induced colorectalcancer mouse models

JMF3086 potently decreased the mRNA expression of proin-flammatory cytokines and chemokines in the colons of AOM/DSS–induced colorectal cancermice, and themRNAexpression ofCOX-II, which is an important tumormarker for colorectal cancer,as well as the proliferation marker cyclin D1 (Fig. 5A). JMF3086also reduced the intracolonic infiltration of inflammatory cells,such as macrophages and neutrophils (F4/80 and Ly-6G), anddecreased COX-II expression around the sites of tumors (Fig. 5Band Supplementary Fig. S5). The efficacy of JMF3086 wassuperior to the combination of lovastatin and SAHA(Figs. 5B, 4A and B). In addition, JMF3086 did not induce in vivoapoptosis despite of protecting AOM/DSS–induced colorectalcancer (Fig. 5C and D).

JMF3086 treatment did not exhibit any toxicity based onbiochemical examinations and H&E staining of various organs(Supplementary Fig. S6A and S6B). However, combination oflovastatin and SAHA increased serum creatinine levels andreduced the concentration of albumin (Supplementary Fig.S6A), indicating renal toxicity.

JMF3086 prevents cancer stemness and endotoxin release inAOM/DSS–induced colorectal cancer mouse models

It has been suggested that chronic inflammation causes cryptdamage and regeneration, resulting in stem cell expansion andleading to colorectal tumorigenesis (17, 26). CD166, EpCAM,CD44, and ALDH1 are putative cell-surface markers associatedwith colorectal CSCs (26, 27). The mRNA expression of these





markers was found to be increased in AOM/DSS–induced colo-rectal cancer mice (Fig. 6A). JMF3086 prevented the induction ofthese markers, whereas statins and SAHA were ineffective orweaker (Fig. 6A).

The microbiota and the immune system mutually interact tomaintainhomeostasis in the intestine.However, endotoxin releasedfrom the microbiota can alter this balance and promote chronicinflammation and lead to intestinal tumor development (28). To

AN

o. o

f tum

ors/

mou

se

Sum

of t

umor

di

amet

ers

(mm

)

**

B

Vehicle (oil)

LOVA50 mg/kg

JMF308650 mg/kg

JMF308625 mg/kg

AOM+DSS (week 8)

Control

H&

E

Vehicle(oil)

LOVA50 mg/kg

JMF308650 mg/kg

JMF308625 mg/kgControl

AOM+DSS (week 8)

Vehicle, n = 10JMF3086 25 mg/kg, n = 6JMF3086 50 mg/kg, n = 12LOVA 50 mg/kg, n = 11SAHA 50 mg/kg, n = 5LOVA + SAHA, n = 5

SAHA50 mg/kg

LOVA+SAHA

** ***

SAHA50 mg/kg

LOVA+ SAHA

* ****

** ****

0 20 40 60 80 100 120 140–2

0

2

4

6

8

10

50 100 150 200 250 300–5

0

5

10

15

20

0

40

80

120

160

1 2 3 4 5 6 7

*

C

HD

AC

Act

ivity

(%

of c

ontr

ol)

Vehicle, n = 8



Control, n = 6

HM

GR

Act

ivity

(%

of c

ontr

ol)

SAHA 50 mg/kg, n = 6LOVA + SAHA, n = 5

0

50

100

150

1 2 3 4 5 6 7

##

******

HMGR

**

##

****

HDAC

****

D

No.

of t

umor

s/m

ouse

R = 0.979 P < 0.001

AOM+DSS (week 8)

0 20 40 60 80 100 120 140–5

0

5

10

15

20

R = 0.978 P < 0.001

R = 0.992P < 0.001

HDAC Activity(% of control)

HDAC and HMGR activity(% of control)

Sum

of t

umor

di

amet

ers

(mm

)

40 60 80 100 120 140 160–4–202468

1012

R = 0.938 P < 0.001

50 100 150 200 250 300–2

0

2

4

6

8

10R = 0.999P < 0.001

40 60 80 100 120 140 160–5

0

5

10

15

20

R = 0.943 P < 0.001

HMGR Activity (% of control)

20

15

10

5

0

20

15

10

5

0

Figure 4.

Preventive effect of JMF3086 on tumor growth in AOM/DSS mouse models. A, representative terminal colons (top) and number of tumors per mouse and total tumorsize (bottom) are shown. � , P < 0.05; ��, P < 0.01 versus vehicle. B, representative of terminal colon sections counterstained with H&E, and high-magnificationimages of the black-boxed area are presented in the bottom row. Scale bar, 250 mm. C, HMGR and HDAC activities in cell lysates from colon tissues are shown. ##, P < 0.01versus control; � , P < 0.05; and �� , P < 0.01 versus vehicle. D, correlations between HDAC activity, HMGR activity, and the number of tumors and tumor sizes are shown.

Wei et al.




05

1015202530

1 2 3 4 5 6 7

A

Rel

ativ

e m

RN

A le

vel

CXCL1 CXCL2

##

****

**

CCL2 COX-II Cyclin D1

0306090

120150

1 2 3 4 5 6 7 0

200

400

600

800

1 2 3 4 5 6 7

020406080

100120

1 2 3 4 5 6 7 050

100150200250

1 2 3 4 5 6 7 0

4

8

12

16

1 2 3 4 5 6 7 05

10152025

1 2 3 4 5 6 7 012345678

1 2 3 4 5 6 7

0

10

20

30

40

1 2 3 4 5 6 7

****

##

****

** ****

##

****

** ****

##

**** **

**

##

****

** ****

##

****

** ****

##

**** **

**

##

***

****

**

##

**** **

**

Vehicle, n = 8



Control, n = 6

SAHA 50 mg/kg, n = 6LOVA+SAHA, n = 5

Rel

ativ

e m

RN

A le

vel

Ly-6

GF4

/80

CO

X-II

BVehicle

(oil)LOVA

50 mg/kgJMF308650 mg/kg

JMF308625 mg/kg

AOM+DSS (week 8)Control SAHA

50 mg/kgLOVA

+ SAHA

F4/80

Vehicle, n = 3



Control, n = 3

COX-IILy-6G

0

20

40

60

80

control vehi cle JMF3086-25 JMF3086-50 LOVA-50 SAHA-50 LOVA+SAHA

0

20

40

60

80

100

control vehi cle JMF3086- 6803FMJ52 -50 LOVA-50 SAHA-50 LOVA+SAHA

0

20

40

60

80

control vehi cle JMF3086-25 JMF3086-50 LOVA-50 SAHA-50 LOVA+SAHA


Mea

n D

AB

inte

nsity

##

**** **

**

##

** ****

**

##

****

**

**

Mea

n D

AB

inte

nsity

M

ean

DA

B in

tens

ity

C

Vehicle (oil)

LOVA50 mg/kg

JMF308650 mg/kg

JMF308625 mg/kg

AOM+DSS (week 8)

Control

TUNEL

D AOM/DSS (week 8)

#1 #1 #2 #1 #2 #1 #2 #1 #2Cleaved PARP

Cleaved caspase-3

Pro-PARP

Pro-caspase 3

BAX

SAHA50 mg/kg

LOVA+ SAHA

25 kDa

35 kDa

25 kDa15 kDa

100 kDa130 kDa

70 kDa

Actin40 kDa

TNFa IFNgIL6IL1b

Figure 5.

Preventive effect of JMF3086 on immune responses in AOM/DSSmousemodels.A, the level of various cytokines and chemokines in colon culture supernatants wasseen. Error bars, SE. �� , P < 0.01 versus vehicle, ##, P < 0.01 versus normal mouse tissue lysates (control); two-sided Student t test. B, colon sections wereimmunostained with anti-F4/80, anti-Ly-6G, and anti-COX-II antibodies, and one representative experiment of three was presented (left). Scale bar, 250 mm.Right, quantification of IHC performed by TissueFAXS software. Error bars, SD. ##, P < 0.01 versus normal mouse paraffin sections (control); �, P < 0.05; and�� , P < 0.01 versus vehicle; two-sided Student t test. C, colon tissues were taken from colorectal cancer mice for prevention experiments, and TUNEL assaywas employed to detect apoptotic cells. Higher magnification views of cells are indicated by arrows, and one representative experiment of three is presented(original magnification: �400, �1,000). D, effect of JMF3086 on apoptotic proteins in colon tissues. Total cell lysates were prepared and the expressionsof PARP, caspase-3, BAX, and b-actin were analyzed by Western blot analysis.





evaluate whether JMF3086maintained gut homeostasis, endotoxinlevel in the serumofAOM/DSS–induced colorectal cancermicewasdetected. High level of endotoxin was detected in AOM/DSS–treated group, and this effect was prevented by JMF3086 with theextent correlated with the reductions in tumor number (Fig. 6B).

DiscussionChemoprevention is a rational and appealing approach to

reduce the risk of cancer by using pharmacologic means, suchas aspirin for colorectal cancer and tamoxifen for breast cancer

02468

1012

1 2 3 4 5 6 7

CD44

A

EpCAMCD166 ALDH1

AOM/DSS (week 8)

Rel

ativ

e m

RN

A le

vel

Vehicle, n = 7



Control, n = 7


0

4

8

12

16

1 2 3 4 5 6 7 02468

1012141618

1 2 3 4 5 6 70

5

10

15

20

25

1 2 3 4 5 6 7

****

##

***

** ****

##

**** ****

##

**** ****

##

****

BVehicle

(oil)JMF30865 mg/kg

JMF30861 mg/kg

AOM/DSS (week 8)

JMF308625 mg/kg

JMF308610 mg/kgControl

Inflammation–dysplasia–adenocarcinoma

prevent colorectal carcinogenesis throughStatin hydroxamatesInhibiting inflammatory cytokines and chemokines1.

D1Attenuating COX-II and cyclin2.Reducing infiltration of macrophages and neutrophils3.

(LPS)Inhibiting the release of endotoxin4.Reducing cancer stemness5.

C

14

12

10

8

6

4

2

0

Endo

toxi

n (E

U/m

L)

AOM/DSS (week 8)

1 5 10 25

Contro

lVe

hicle

JMF3086 (mg/kg)

Figure 6.

Preventive effect of JMF3086 on cancer stemness and endotoxin release in AOM/DSS mouse models. A, the mRNA expressions of colorectal CSC markersin colon tissues from 8-week-old AOM/DSS colorectal cancer mice of prevention model are shown. ##, P < 0.01 versus normal mouse tissue lysates (control);� , P < 0.05; and �� , P < 0.01 versus vehicle. B, gross pictures of terminal colons are shown, and arrowhead indicates macroscopic lesions (left). Scale bars,5 mm. Colon sections were counterstained with H&E. Scale bars, 250 mm. Endotoxin concentration (EU/mL) of serum from AOM/DSS–induced colorectalcancer mouse was measured by limulus amebocyte lysate (LAL) assay (right). Error bars, SE. � , P < 0.05; ��, P < 0.01 versus vehicle; ##, P < 0.01 versus normalmouse serum (control); two-sided Student t test. C, connection between inflammation and carcinogenesis and prevention by statin hydroxamates. The oraladministration of statin hydroxamate prevented AOM/DSS–induced colorectal cancer in mice by inhibiting inflammation. Statin hydroxamate also inhibited therelease of endotoxin in vivo. Furthermore, the stemness of colorectal cancer was inhibited in vivo as well. These data provided compelling evidence thatstatin hydroxamate showed significant efficacy to prevent colorectal cancer in preclinical models.


Wei et al.



(29, 30). Aspirin has been reported to prevent carcinogenesis ofcolorectal cancer in ApcMin/þ mice and prevent 44% tumor for-mation at a dosage of 200 mg/kg through attenuating inflamma-tion (31, 32). However, its clinical use for chemoprevention isprohibited due to a significant incidence of gastrointestinal upsetand bleeding. Epigeneticmodulators, such asHDAC2overexpres-sion, were associated with colorectal cancer progression, and aselective HDAC2 inhibitor was reported to prevent colorectalcancer tumorigenesis in both AOM/DSS mouse models andApcMin/þ mice (33). A recent study shows that gut microbiotacould ferment dietary fiber into butyrate, a class I and II HDACinhibitor, to exert chemopreventive effect on colorectal cancer ingnotobiotic mouse models (34), suggesting that HDACs arepotential targets for colorectal cancer prevention. In addition,epidemiology study suggests that statins attenuated the risk ofcolorectal cancer (35). In this study, our polypharmacologicalsmall molecule, statin hydroxamate (JMF3086), dually inhibitsHDACs and HMGR exerting anti-inflammatory effect to preventcolitis and colitis-associated colorectal cancer. HMGR and HDACactivities in colon lysates of AOM/DSS–induced colorectal cancerwere increased and inhibited by JMF, indicating that both inhibi-tions contributed to its chemoprevention. The advantage of JMFbeing superior to lovastatin plus SAHA is like a two-in-oneantibody with superior inhibitory activity, compared with twomonospecific antibodies (36). Additional mechanisms beyondthe inhibitions onHDAC andHMGR activities or due to differentpharmacokinetic property are also possible and awaited forfurther investigation. However, we reveal a new strategy forcolorectal cancer chemoprevention.

IBD is an inflammatory disorder of the gastrointestinal tract(6). Epidemiologic studies suggest that the incidence and prev-alence of IBD are increasing around the world. However, the exactetiology remains unknown and disease pathogenesis is not fullyunderstood. DSS-induced colitis has been shown to be similar tohuman IBD inmultiple aspects andhasbecome an important toolto investigate its pathophysiologic mechanisms and immunolog-ic processes (37). DSS-induced colitis is triggered by disruptingthe colon epithelial barrier, allowing intestinal bacteria to pen-etrate the injured mucosa and induce mucosal inflammation,which is characterized by increased infiltration of inflammatorycells and an excessive production of proinflammatory cytokines/chemokines, leading to colitis exacerbation (38). Therefore, inhi-bition of inflammation is a potential strategy to prevent cancerinitiation. In this study, we found JMF3086 could prevent andtreat acute inflammation in the DSS-induced colitis mouse mod-els by inhibiting the release of proinflammatory cytokines/che-mokines and the infiltration of neutrophils into colitis regions.AOM in conjunction with the additional inflammatory stimulusof DSS results in tumor development that is restricted to the colonin mice (12). This model is widely utilized to recapitulate humancolorectal cancer through inflammation and colon ulceration,similar to what is observed in patients (39). JMF3086 is demon-strated to prevent AOM/DSS colorectal cancer in this study,indicating that both initiation and progression of colorectalcancer are inhibited by JMF3086.

Tumors are tightly connected with microbiota and immunesystem (40). The link betweenmicrobiota and colorectal cancer inpatients and mice has been studied and found that germ-freeanimals showed fewer tumors compared with those of normalmicrobiota. Antibiotic cocktail depleting the intestinal micro-biota inmice reduced tumor growth in liver and colon, suggesting

that microbiota promotes tumor progression (41). Specifically,Fusobacterium nucleatum compared with healthy people is morecommon in the gut of IBD patients to promote colorectal cancer(42), and clinical isolate of F. nucleatum promoted carcinogenesisin ApcMin/þ mice (43). The gut microbiota modulates carcino-genesis through three mechanisms (28). First, dysbiosis inducesbacterial translocation, leading to induction of inflammationmediated by microorganism-associated molecular patterns andToll-like receptors in several cell types (41). Intestinal inflamma-tion promotes colorectal cancer development through release ofproinflammatory cytokines/chemokines and the infiltration ofmacrophages and neutrophils into tumor regions (28, 44). Bac-terial translocation was detected at tumor sites in ApcMin/þ mice,and antibiotic eradication reduced colorectal cancer development(45). Second, microbiota-derived metabolites could induce DNAdamage, genotoxicity, and chromosome instability to inducebarrier failure of gastrointestinal tract, which also trigger colorec-tal cancer carcinogenesis. For example, GNB-produced LPS andcytolethal distending toxin directly trigger double-strand DNAdamage responses, leading to barrier disrupt. Third, microbiotacould convert bile acid in host into secondarymetabolites, such asdeoxycholic acid, which in turn promotes the development ofhepatocellular carcinoma and colorectal cancer (46, 47). In thisstudy, increasedmRNAexpressions of proinflammatory cytokinesand chemokines, infiltrations of macrophage (F4/80) and neu-trophil (Ly-6G), as well as increased serum endotoxin (LPS) levelwere seen in AOM/DSS colorectal cancer mousemodels. All theseevents were prevented by JMF3086, suggesting that JMF3086might prevent colorectal cancer development through modulat-ing gut microbiota–induced bacterial translocation and barrierfailure of gastrointestinal tract. It is important to find a reliablebiomarker to detect the effect of chemopreventive agents. Serumendotoxin level might serve as a potential biomarker to detect theprevention of JMF3086 in colorectal cancer.

The collection of bacteria, viruses, and fungi that live in thehumanbodywas knownas the "microbiome" (48). It is estimatedthat less than 1% of all microorganisms in the natural world havebeen identified (49). It was largely unknown and regarded as ablack box. Recently, the advent of new technologies, includinghigh-throughput DNA sequencing and bioinformatics, hasincreased the capacity and launched a revolution in the micro-biome (40). In this study, we found that JMF3086 preventedendotoxin release in AOM/DSS–induced colorectal cancermousemodels as well as the treatmentmodel of DSS-induced colitis. It ispossible that JMF3086 acts throughmodulating the population ofgut microbiota. High-throughput microbial 16S rRNA genesequencing combined with mouse models is awaited to discoverfunctional genes of microbiota impacted by JMF3086.

Colorectal CSCs have been reported to initiate colorectal cancerand to play an important role in the genesis, maintenance,recurrence, and metastasis of cancer (50, 51). The expression ofcolorectal CSC markers, CD166, EpCAM, CD44, and ALDH1, inAOM/DSS–induced colorectal cancer mouse models was pre-vented by JMF3086, indicating that inhibition of CSCs mightalso contribute to its chemoprevention on colorectal cancer.Statins and HDAC inhibitors have been reported to reduce thestemness of colorectal CSCs (52–54).

In conclusion, our findings indicated that JMF3086, which is apotent dual inhibitor targeting HDACs and HMGR, has potentialbenefit and is a promising lead for the prevention of colitis-associated colorectal cancer.





Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T.-T. Wei, C.-C. ChenDevelopment of methodology: T.-T. Wei, Y.-T. Lin, R.-Y. TsengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.-T. Wei, Y.-T. Lin, R.-Y. TsengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.-T. Wei, Y.-T. Lin, R.-Y. Tseng, C.-T. ShunWriting, review, and/or revision of the manuscript: T.-T. Wei, Y.-C. Lin,M.-S. Wu, C.-C. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T.-T. Wei, M.-S. Wu, J.-M. Fang, C.-C. Chen

Study supervision: C.-C. ChenOther (obtained funding): C.-C. Chen

Grant SupportThis work was supported by a research grant from the National Science

Council of Taiwan and the Institute of Biomedical Sciences, Academia Sinica(IBMS-CRC103-P02).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 3, 2015; revised March 19, 2016; accepted April 11, 2016;published online July 15, 2016.

References1. Sporn MB. Approaches to prevention of epithelial cancer during the

preneoplastic period. Cancer Res 1976;36:2699–702.2. Burn J, Gerdes AM, Macrae F, Mecklin JP, Moeslein G, Olschwang S, et al.

Long-term effect of aspirin on cancer risk in carriers of hereditary colorectalcancer: an analysis from the CAPP2 randomised controlled trial. Lancet2011;378:2081–7.

3. Fink SP, Yamauchi M, Nishihara R, Jung S, Kuchiba A, Wu K, et al. Aspirinand the risk of colorectal cancer in relation to the expression of 15-hydroxyprostaglandin dehydrogenase (HPGD). Sci Transl Med 2014;6:233re2.

4. Terzic J, Grivennikov S, Karin E, Karin M. Inflammation and colon cancer.Gastroenterology 2010;138:2101–14.e5.

5. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, et al. IKKbetalinks inflammation and tumorigenesis in a mouse model of colitis-asso-ciated cancer. Cell 2004;118:285–96.

6. Ullman TA, Itzkowitz SH. Intestinal inflammation and cancer. Gastroen-terology 2011;140:1807–16.

7. Moossavi S, Bishehsari F. Inflammation in sporadic colorectal cancer.Arch Iran Med 2012;15:166–70.

8. Bardou M, Barkun AN, Martel M. Obesity and colorectal cancer. Gut2013;62:933–47.

9. Tang J, Sharif O, Pai C, Silverman AL. Mesalamine protects against colorectalcancer in inflammatory bowel disease. Dig Dis Sci 2010;55:1696–703.

10. van Staa TP, Card T, Logan RF, Leufkens HG. 5-Aminosalicylate use andcolorectal cancer risk in inflammatory bowel disease: a large epidemio-logical study. Gut 2005;54:1573–8.

11. ArumugamM, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, et al.Enterotypes of the human gut microbiome. Nature 2011;473:174–80.

12. Chen GY, Nunez G. Inflammasomes in intestinal inflammation andcancer. Gastroenterology 2011;141:1986–99.

13. Round JL, Mazmanian SK. The gut microbiota shapes intestinal immuneresponses during health and disease. Nat Rev Immunol 2009;9:313–23.

14. Sommer F, Backhed F. The gut microbiota–masters of host developmentand physiology. Nat Rev Microbiol 2013;11:227–38.

15. Macarthur M, Hold GL, El-Omar EM. Inflammation and Cancer II. Role ofchronic inflammation and cytokine gene polymorphisms in the patho-genesis of gastrointestinal malignancy. Am J Physiol Gastrointest LiverPhysiol 2004;286:G515–20.

16. Hull M, Lagergren J. Obesity and colorectal cancer. Gut 2014;63:205.17. Carpentino JE, HynesMJ, AppelmanHD, Zheng T, Steindler DA, Scott EW,

et al. Aldehyde dehydrogenase-expressing colon stem cells contribute totumorigenesis in the transition from colitis to cancer. Cancer Res2009;69:8208–15.

18. Kapur NK. Rosuvastatin: a highly potent statin for the prevention andmanagement of coronary artery disease. Expert Rev Cardiovasc Ther 2007;5:161–75.

19. Ward MM. The JUPITER study: statins for the primary prevention ofcardiovascular events in patients with inflammatory rheumatic diseases?F1000 Med Rep 2009;1:35.

20. Hawk E, Viner JL. Statins and cancer–beyond the "one drug, one disease"model. N Engl J Med 2005;352:2238–9.

21. Lin YC, Lin JH,ChouCW,Chang YF, Yeh SH,ChenCC. Statins increase p21through inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2. Cancer Res 2008;68:2375–83.

22. West AC, Johnstone RW. New and emerging HDAC inhibitors for cancertreatment. J Clin Invest 2014;124:30–9.

23. Chen JB,Chern TR,Wei TT, ChenCC, Lin JH, Fang JM.Design and synthesisof dual-action inhibitors targeting histone deacetylases and 3-hydroxy-3-methylglutaryl coenzyme A reductase for cancer treatment. J Med Chem2013;56:3645–55.

24. Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors incancer, neurological diseases and immune disorders. Nat Rev Drug Discov2014;13:673–91.

25. Neurath MF. Cytokines in inflammatory bowel disease. Nat Rev Immunol2014;14:329–42.

26. Huang EH,WichaMS. Colon cancer stem cells: implications for preventionand therapy. Trends Mol Med 2008;14:503–9.

27. LaBargeMA, Bissell MJ. Is CD133 amarker of metastatic colon cancer stemcells? J Clin Invest 2008;118:2021–4.

28. Gagliani N, Hu B, Huber S, Elinav E, Flavell RA. The fire within: microbesinflame tumors. Cell 2014;157:776–83.

29. Reddy BS, Hirose Y, Lubet R, Steele V, Kelloff G, Paulson S, et al. Chemo-prevention of colon cancer by specific cyclooxygenase-2 inhibitor, cele-coxib, administered during different stages of carcinogenesis. Cancer Res2000;60:293–7.

30. Fisher B, Costantino JP, Wickerham DL, Cecchini RS, Cronin WM, Robi-doux A, et al. Tamoxifen for the prevention of breast cancer: current statusof theNational Surgical Adjuvant Breast and Bowel Project P-1 study. J NatlCancer Inst 2005;97:1652–62.

31. Bosetti C, Rosato V,Gallus S, Cuzick J, La VecchiaC. Aspirin and cancer risk:a quantitative review to 2011. Ann Oncol 2012;23:1403–15.

32. Mahmoud NN, Dannenberg AJ, Mestre J, Bilinski RT, Churchill MR,Martucci C, et al. Aspirin prevents tumors in a murine model of familialadenomatous polyposis. Surgery 1998;124:225–31.

33. Ravillah D, Mohammed A, Qian L, Brewer M, Zhang Y, Biddick L, et al.Chemopreventive effects of an HDAC2-selective inhibitor on rat coloncarcinogenesis and APCmin/þmouse intestinal tumorigenesis. J Pharma-col Exp Ther 2014;348:59–68.

34. Donohoe DR, Holley D, Collins LB, Montgomery SA, Whitmore AC,Hillhouse A, et al. A gnotobiotic mouse model demonstrates that dietaryfiber protects against colorectal tumorigenesis in a microbiota- and buty-rate-dependent manner. Cancer Discov 2014;4:1387–97.

35. Poynter JN, Gruber SB, Higgins PD, Almog R, Bonner JD, Rennert HS,et al. Statins and the risk of colorectal cancer. N Engl J Med 2005;352:2184–92.

36. Schaefer G, Haber L, Crocker LM, Shia S, Shao L, DowbenkoD, et al. A two-in-one antibody against HER3 and EGFR has superior inhibitory activitycompared with monospecific antibodies. Cancer Cell 2011;20:472–86.

37. Mizoguchi A. Animal models of inflammatory bowel disease. Prog MolBiol Transl Sci 2012;105:263–320.

38. Maloy KJ, Powrie F. Intestinal homeostasis and its breakdown in inflam-matory bowel disease. Nature 2011;474:298–306.

Wei et al.




39. Alex P, Zachos NC, Nguyen T, Gonzales L, Chen TE, Conklin LS, et al.Distinct cytokine patterns identified from multiplex profiles of murineDSS and TNBS-induced colitis. Inflamm Bowel Dis 2009;15:341–52.

40. Blaser MJ. The microbiome revolution. J Clin Invest 2014;124:4162–5.41. Schwabe RF, Jobin C. The microbiome and cancer. Nat Rev Cancer

2013;13:800–12.42. Allen-Vercoe E, Strauss J, Chadee K. Fusobacterium nucleatum: an emerg-

ing gut pathogen? Gut Microbes 2011;2:294–8.43. Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M,

et al. Fusobacterium nucleatum potentiates intestinal tumorigenesis andmodulates the tumor-immune microenvironment. Cell Host Microbe2013;14:207–15.

44. Galdiero MR, Bonavita E, Barajon I, Garlanda C, Mantovani A, Jaillon S.Tumor associated macrophages and neutrophils in cancer. Immunobio-logy 2013;218:1402–10.

45. Grivennikov SI, Wang K, Mucida D, Stewart CA, Schnabl B, Jauch D, et al.Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth. Nature 2012;491:254–8.

46. Nesic D, Hsu Y, Stebbins CE. Assembly and function of a bacterialgenotoxin. Nature 2004;429:429–33.

47. BernsteinC,HolubecH, Bhattacharyya AK,NguyenH, PayneCM,Zaitlin B,et al. Carcinogenicity of deoxycholate, a secondary bile acid. Arch Toxicol2011;85:863–71.

48. Lederberg J. Infectious history. Science 2000;288:287–93.49. Fuhrman JA. Microbial community structure and its functional implica-

tions. Nature 2009;459:193–9.50. Eyler CE, Rich JN. Survival of the fittest: cancer stem cells in therapeutic

resistance and angiogenesis. J Clin Oncol 2008;26:2839–45.51. PignalosaD,DuranteM.Overcoming resistance of cancer stem cells. Lancet

Oncol 2012;13:e187–8.52. Gauthaman K, Fong CY, Bongso A. Statins, stem cells, and cancer. J Cell

Biochem 2009;106:975–83.53. Kodach LL, Jacobs RJ, Voorneveld PW,Wildenberg ME, Verspaget HW, van

Wezel T, et al. Statins augment the chemosensitivity of colorectal cancercells inducing epigenetic reprogramming and reducing colorectal cancercell 'stemness' via the bone morphogenetic protein pathway. Gut2011;60:1544–53.

54. Sikandar S,DizonD, ShenX, Li Z, Besterman J, Lipkin SM. The class IHDACinhibitor MGCD0103 induces cell cycle arrest and apoptosis in coloncancer initiating cells by upregulating Dickkopf-1 and non-canonical Wntsignaling. Oncotarget 2010;1:596–605.





Correction

Correction: Prevention of Colitis andColitis-Associated Colorectal Cancer by aNovel Polypharmacological HistoneDeacetylase Inhibitor

In this article (ClinCancer Res 2016;22:4158–69),whichwaspublished in theAugust15, 2016, issue of Clinical Cancer Research (1), the corresponding author alerted thejournal to mismatches between the images and their enlargements in several panelsin Fig. 4B. The conclusions put forth in the article remain unchanged. The correctedversion of Fig. 4B is provided below. The authors regret this error.

Reference1. Wei T, Lin Y, TsengR, ShunC, LinY,WuM, et al. Preventionof colitis and colitis-associated colorectal

cancer by a novel polypharmacological histone deacetylase inhibitor. Clin Cancer Res 2016;22:4158–69.

Published online January 16, 2018.doi: 10.1158/1078-0432.CCR-17-2859�2018 American Association for Cancer Research.

Figure 4B.

ClinicalCancerResearch

www.aacrjournals.org 499

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-2859&domain=pdf&date_stamp=2017-12-29

2016;22:4158-4169. Clin Cancer Res Tzu-Tang Wei, Yi-Ting Lin, Ruo-Yu Tseng, et al. Novel Polypharmacological Histone Deacetylase InhibitorPrevention of Colitis and Colitis-Associated Colorectal Cancer by a

Updated version

http://clincancerres.aacrjournals.org/content/22/16/4158

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2016/08/26/1078-0432.CCR-15-2379.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/22/16/4158.full#ref-list-1

This article cites 54 articles, 13 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/22/16/4158.full#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/22/16/4158To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/content/suppl/2016/08/26/1078-0432.CCR-15-2379.DC1

http://clincancerres.aacrjournals.org/content/22/16/4158.full#ref-list-1

http://clincancerres.aacrjournals.org/content/22/16/4158.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Prevention of Colitis and Colitis-Associated ... · the association between chronic inﬂammation (colitis) and colorectal cancer (4, 6). In addition, both sporadic and herita-ble