Author Manuscript Published OnlineFirst on August 19, 2020; DOI: 10.1158/0008-5472.CAN-20-0469 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ARC is a critical protector against inflammatory bowel disease (IBD) and IBD-associated colorectal tumorigenesis

Qiushi Wang1*, Tianshun Zhang1*, Xiaoyu Chang1, Do Young Lim1, Keke Wang1, Ruihua Bai1,2,

Ting Wang1, Joohyun Ryu1, Hanyong Chen1, Ke Yao1, Wei-Ya Ma1, Lisa A. Boardman3, Ann M.

Bode1, Zigang Dong1,4+

1The Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN 55912

2The Henan Tumor Hospital, No.127 Dongming Road, Zhengzhou, Henan, China, 450000

3Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, 55905, USA,

4Department of Pathophysiology, School of Basic Medical Sciences. College of Medicine.

Zhengzhou University, Henan, 450001, China

*Qiushi Wang and Tianshun Zhang have contributed equally to this work

Corresponding Author:

+Address correspondence to Zigang Dong, No.100 Science Avenue, Zhengzhou City, Henan

Province, China. Postcode: 450001. Telephone: +86-371-66658803; Email: [email protected]

Conflicts of Interest: The authors declare no potential conflicts of interest

Running title: ARC for IBD-associated colorectal tumorigenesis 1

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Author contributions:

QW and TZ contributed equally to the manuscript and designed the experiments, performed experiments, analyzed and interpreted data, prepared figures and wrote the manuscript; XC,

DYL and KW conducted experiment and analyzed data; RB performed pathological identification; TW supported for the animal studies; JR performed mass spectrometry analysis.

HC contributed to computational analysis; KY and WM assisted in establishing experimental methods. LAB collected and prepared the human samples; AMB contributed to review and revision of manuscript. ZD contributed to study supervision, experimental design, data discussion, and revision of manuscript.

Keywords: ARC, TRAF6, ubiquitination, inflammatory bowel disease (IBD), colorectal tumorigenesis

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Abstract

The key functional molecules involved in inflammatory bowel disease (IBD) and IBD-induced colorectal tumorigenesis remain unclear. In this study, we found that the apoptosis repressor with caspase recruitment domain (ARC) plays critical roles in IBD. ARC-deficient mice exhibited substantially higher susceptibility to dextran sulfate sodium (DSS)-induced IBD compared to wild-type (WT) mice. The inflammatory burden induced in ARC-deficient conditions was inversely correlated with CCL5 and CXCL5 levels in immune cells, especially

CD4- positive T cells. Pathologically, ARC expression in immune cells was significantly decreased in clinical biopsy specimens from IBD patients compared with normal subjects.

Additionally, ARC levels inversely correlated with CCL5 and CXCL5 levels in human biopsy specimens. ARC interacted with tumor necrosis factor receptor associated factor (TRAF) 6, regulating ubiquitination of TRAF6, which was associated with nuclear factor-kappa B (NF-kB) signaling. Importantly, we identified a novel ubiquitination site at lysine 461, which was critical in the function of ARC in IBD. ARC played a critical role in IBD and IBD-associated colon cancer in a bone marrow transplantation model and AOM/DSS-induced colitis cancer mouse models. Overall, these findings reveal that ARC is critically involved in the maintenance of intestinal homeostasis and protection against IBD through its ubiquitination of TRAF6 and subsequent modulation of NF-κB activation in T cells.

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Introduction

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s

disease (CD), is a chronic inflammatory disease of the gastrointestinal tract (1,2). IBD can cause

various complications, such as abscesses, fistulas, colitis-associated neoplasias and cancer (3).

Approximately 1.6 million patients suffer from the disease in the United States and 2.5 to 3

million in Europe (4,5). IBD is associated with an immunological imbalance of the intestinal mucosa, mainly related to cells in the adaptive immune system leading to chronic inflammation

conditions in patients. The pathophysiological mechanisms of IBD are still not clear, even

though these diseases were discovered several decades ago (6-8).

The protein apoptosis repressor with caspase recruitment domain (ARC), also referred to as

Nol3, plays an important role in suppressing apoptotic responses (9,10). ARC was believed to

exert its function through multiple protein-protein interactions and transcriptional regulation (11).

ARC was primarily discovered as an endogenous inhibitor of cell death and is highly expressed

in cardiomyocytes, skeletal muscle cells, and neurons under physiological conditions (12-14). It

was independently identified in later studies as having other functions, including

post-translational modifications like phosphorylation, calcium binding, and ubiquitination

(15,16). ARC has increased expression in solid tumors and in patients with acute myeloid

leukemia to mediate the response of cells to the induction of pharmacological apoptosis (17-20).

Intriguingly, studies also revealed that ARC might perform its function as a tumor suppressor in

renal cell carcinoma cells and myeloid tumors (21,22). These findings suggest dual roles for

ARC in oncogenesis that may be cell type-dependent. The CARD family participates in the 4

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regulation of apoptosis, inflammation, and NF-κB signaling pathway activation. However, the function of ARC in the inflammation reaction is still not clear (23,24). Recently, ARC was shown to play a pivotal role in the pathogenesis of acute kidney injury and ARC knockout markedly accelerated the expression levels of inflammatory factors (25). Moreover, ARC has been reported to suppress NF-κB pathway activation and to interact directly with p53 to disrupt its transcriptional activity (23,26). These findings suggest that ARC plays potential roles in the inflammatory response and cancer development. Additional, ARC is high expression in almost all primary colon cancers compared with corresponding controls, suggesting that ARC is a novel marker for human colon cancer (27). However, the function of ARC in IBD and colon cancer has not yet been assessed.

In this study, we examined the function of ARC in IBD and IBD-associated colon cancer.

We demonstrated a role for ARC in regulation of inflammatory response in an ARC-deficient mouse model and clinical biopsy specimens. We identified ARC as a protector for IBD in immune cells and also aimed to ascertain the mechanisms of ARC related to the inflammatory response in IBD development. Furthermore, the bone marrow transplantation and AOM/DSS mouse model were used to study the effect of ARC on IBD and IBD-associated colorectal tumorigenesis. Our findings uncover a critical role for ARC in IBD development and give rise to a potential new strategy for IBD therapy.

Materials and Methods

Reagents and antibodies 5

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Cell culture media, gentamicin, penicillin, and L-glutamine were all obtained from

Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Gemini Bio-Products (West

Sacramento, CA) and Tris, NaCl, and SDS for molecular biology and buffer preparation were

purchased from Sigma-Aldrich (St. Louis, MO). Antibodies to detect -actin (sc-47778), NF-B

(p50) (sc-7178), Lamin B (sc-6216), CD4 (sc-13573), ARC (sc-11435), ENA-78 (sc-377026),

RANTES (sc-514019), TRAF1 (sc-271683), TRAF4 (sc-10776), TRAF5 (sc-74502) and TRAF6

(sc-8409) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). His-HRP (R94125) were

from Invitrogen. Anti-HA (901503) was obtained from Covance (Emeryville, CA) and

anti-HA-HRP (12013819001) was purchased from Roche (Indianapolis, IN). Anti-Flag (F-3165)

was from Millipore sigma. Anti-CCL5 (ab9679) and anti-CXCL5 (ab9802) were purchased from

Abcam (Cambridge, MA). NF-B (p65) (#3034), TRAF2 (#14712), PE-ARC (#89210) and

TRAF3 (#4729T) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

PE-CCL5 (149104), FITC-CD4 (557307), FITC-CD8 (553030), FITC-CD49b (561067) and

FITC-CD19 (557398) were purchased from Biolegend (San Diego, CA).

Construction of expression vectors

Expression constructs, including ARC and HA-Lys-63-ubiquitin were obtained from

Addgene (Cambridge, MA). The Packaging vector, pcDNA4/His max vector, was obtained from

Invitrogen (Carlsbad, CA). To construct His-tagged expression vector of ARC, we amplified the

DNA sequences corresponding to HA-ARC by PCR and the HotStarTaq Master Mix Kit (Qiagen,

Chatsworth, CA) was used. We designed specific primers for His-ARC F:5’ AT GGATCC 6

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ATGGGCAACGCGCAGGAG 3’, R: 5’ AT CTCGAG CTATCCAGCATGGGCGGG 3’. The

His-ARC PCR product was digested with BamH I/Xho I following instructions provided by the manufacturer, and then inserted into the corresponding sites of pcDNA4/His max (Invitrogen,

Carlsbad, CA) to generate the expression plasmids encoding His-ARC.

Additionally, the lentivirus plasmids shARC (#1, V2LHS_47055; #2 V2LHS_47054) were purchased from GE Healthcare Dharmacon (OpenBioSystem). The pLKO.1-puro Non-Target shRNA Control Plasmid DNA (shNT) was purchased from Sigma-Aldrich Co. LLC (St. Louis,

MO). All constructs were confirmed by restriction enzyme mapping, DNA sequencing, and

Blast.

Cell culture and transfection

All cells were purchased from American Type Culture Collection (ATCC; Manassas, VA).

The cells were routinely screened to confirm mycoplasma-negative status and to verify the identity of the cells by Short Tandem Repeat (STR) profiling before being frozen. Each vial was thawed and maintained for a maximum of 2 months. Enough frozen vials of each cell line were available to ensure that all cell-based experiments were conducted on cells that had been tested and in culture for 8 weeks or less. Cells were cultured at 37°C in a 5% CO2 humidified incubator following the ATCC protocols. Jurkat and MOLT3 cells were grown in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics. HEK293T cells (stably expressing the SV40 large T antigen in HER293 cells) were purchased from the American Type Culture Collection and cultured at 37°C in a humidified incubator with 5% CO2 in Dulbecco’s modified Eagles’s 7

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medium (DMEM, Corning; Manassas, VA) supplemented with 10% FBS (Corning, Manassas,

VA) and 1% penicillin streptomycin (Gen DEPOT; Barker, TX). When cells reached 60%

confluence, transfection was performed using iMFectin DNA Transfection Reagent (GenDEPOT,

Barker, TX) following the manufacturer’s instructions. The cells were cultured for 36 to 48 h and

were extracted for further analysis.

Immunofluorescence, immunoprecipitation and Western blot analysis

Protein samples were extracted with Nonidet P-40 lysis buffer (50 mM Tris-HCl, pH 8.0,

150 mM NaCl, 0.5% Nonidet P-40 and protease inhibitor mixture). For immunoblotting, 40 g of proteins were detected with specific antibodies and an alkaline phosphatase (AP)-conjugated secondary antibody. Proteins were visualized by chemiluminescence (Amersham Biosciences,

Piscataway, NJ). The immunoprecipitation (IP) assay was performed as described previously

(28). The extractions were precleared with 10 L protein G agarose beads (GenDEPO; Barker,

TX) by rocking for 30 min at 4°C. The precleared supernatant fractions were combined with

fresh protein A/G agarose beads (Santa Cruz, CA) and appropriate 2 g of antibodies were added and rocked overnight at 4°C. The immunoprecipitates were washed four times with the above

lysis buffer. Immunoprecipitates were suspended in SDS sample buffer and subjected to

SDS-PAGE and Western blotting. For IP under denaturing conditions, proteins were extracted

using regular IP lysis buffer plus 1% SDS and heated at 95°C for 5 min. Samples were diluted

ten times by using regular IP lysis buffer before IP. The beads were washed, mixed with SDS

sample buffer, boiled and then resolved by SDS-PAGE. Signals were visualized by 8

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immunoblotting.

Equal amounts of protein were determined using a protein assay kit (Bio-Rad Laboratories,

Hercules, CA). Lysates were resolved by SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) and blocked with 5% nonfat milk for 1 h at room temperature. Blots were probed with appropriate primary antibodies (1:1000) overnight at 4°C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for hybridization. Protein bands were visualized with a chemiluminescence reagent (GE Healthcare Biosciences, Piscataway, NJ).

Inflammation array

Colon tissue lysates from DSS-induced mice were subjected to a mouse inflammation array

C1 (RayBiotech, Peachtree Corners, GA) following the manufacturer’s instructions. Density

scores were obtained using Image J software.

Measurement of ARC, CCL5, and CXCL5 in clinical human samples

The leukocytes (buffy coat) and plasma samples with EDTA from normal subjects or

patients with Crohn’s disease or ulcerative colitis samples were obtained after informed consent

as Mayo IRB protocol #622-00.The baseline demographics are shown in Supplementary Table

1. The measurement of ARC, CCL5, and CXCL5 was performed using immunoassay kits (ARC:

MBS95000636; CCL5: MBS773187; and CXCL5: MBD825081) from MyBioSource (San

Diego, CA) following the manufacturer’s instructions. 9

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Ubiquitination assays

The ubiquitination assay was performed as described previously (28,29). HEK293T cells were transfected with combinations of expression vectors Flag-TRAF6, His-ARC, and

HA-Lys63-ubiquitin. The proteins were extracted using lysis buffer (50 mM Tris-HCl, pH 8.0,

0.5 mM EDTA, 1% SDS, 1 mM DTT) and boiled for 10 min before cellular debris was removed

by centrifugation. The lysates were immunoprecipitated as described above, and IP was

conducted with anti-Flag. Bound proteins were eluted in SDS sample loading buffer and

subjected to immunoblotting.

For the in vitro ubiquitination assay (30), Flag-TRAF6 was co-transfected with HA-ubiquitin

into HEK293T cells. At 24 h later, the transfected cells were stimulated with IL-1β for 15 min.

The cells were harvested using RIPA buffer (100 mM Tris/HCl pH 7.4, 30 mM NaCl, 2.5% sodium deoxycholate, 2 mM EDTA and 2% Nonidet P40) containing protease inhibitor cocktail and NEM (N-ethylmaleimide). The cell lysates were incubated with Flag M2 beads overnight

with rotation at 4oC. After extensive washing with TBS [Tris-buffered saline (25 mM Tris/HCl,

pH 7.4, 150 mM NaCl and 3 mM KCl)], Flag–TRAF6 was eluted with elution buffer [1×PBS

(pH 7.4)] containing 3 × Flag peptide. Then 15 μL of the eluted Flag–TRAF6 was incubated with

purified His-ARC in DUB assay buffer [50 mM HEPEs/NaOH (pH 8.0), 10% glycerol and 3

mM DTT (dithiothreitol)] at 37oC for 4 h. Ubiquitination was analyzed by Western blotting using

anti-His, anti-Flag or anti-HA.

10

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Liquid chromatography-mass spectrometry/mass spectrometry analysis to identify

ubiquitination sites of TRAF6

Flag-TRAF6 was co-expressed with or without His-ARC, along with HA-Ub-K63, into

HEK293T cells. After cell lysis, samples were immunoprecipitated with anti-Flag, then

denatured with urea buffer (7 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl

dimethylammonio) propanesulfonate (CHAPS), reduced with 4 mM dithiothreitol for 1 h at 37°C, and alkylated with 14 mM iodoacetamide for 45 min at room temperature under dark conditions.

Excess iodoacetamide was quenched with excess dithiothreitol to provide a final concentration of

7 mM. Subsequently, the sample was diluted with 25 mM ammonium bicarbonate to ensure less

than 1 M urea content, and digested with trypsin (Promega, Madison, WI) at an enzyme content

of 2% (weight/weight) for 16 h at 37oC. The tryptic peptides were dried by vacuum evaporation

using a speed vacuum and then cleaned up with a Sep-Pak C18 cartridge (Waters, Milford, MA).

The Sciex TripleTOF 5600 system (Sciex, Framingham, MA) coupled with Eksigent 1D+ nano

LC system (Sciex) was used to identify TRAF6 ubiquitination. The mass spectrometry was

automatically calibrated by acquisition of β-galactosidase peptides (25 fmole/μl) in the same

acquisition batch. The raw data were processed and searched with ProteinPliot software (version

4.5; Sciex) using the Paragon algorithm. Proteins were identified by searching the UniProtKB

human database (www..org).

Quantitative real-time PCR

Trizol Reagent (Invitrogen, Carlsbad, CA) was used for total RNA extraction from mouse 11

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colon tissue and Jurkat and MOLT3 cells. The CCL5 and CXCL5 expression was analyzed

with 100 ng of total RNA. Primers included mouse CCL5-specific real-time primers:

F:5’CCATGAAGGTCTCCGCGGCAC-3’; R:5’-CCTAGCTCATCTCCAAAGAG-3’; mouse

CXCL5-specific real-time primers: F:5’-TGGCCCCTTTCACAGAGTAG-3’;

R:5’-CTAAAAACCCGACAGGCATC-3’; and mouse glyceraldehyde 3-phosphate

dehydrogenase-specific real-timer primers: F: 5’-CTTCACCACCATGGAGGAGGC-3’; R:

5’-GGCATGGACTGTGGTCATGAG-3’; human CCL5-specific real-time primesr:

F:5’-GCTGTCATCCTCATTGCTACTG-3’; R:5’-TGGTGTAGAAATACTCCTTGATGTG-3’;

human CXCL5-specific real-time primers: F:5’-TGGACGGTGGAAACAAGG-3’;

R:5’-CTTCCCTGGGTTCAGAGAC-3’; and human a glyceraldehyde 3-phosphate dehydrogenase-specific real-timer primers: F: 5’-GCCCAATACGACCAAATCC-3’; R:

5’-CTCTGCTCCTCCTGTTCGAC-3’. Quantitative one-step real-time PCR using the TaqMan

RNA-to-CT 1-step kit (Applied Biosystems, Foster City, CA) was usedfollowing the

manufacturer’s suggested protocols. The CT values of CCL5 and CXCL5 gene expression were

normalized with the CT values of glyceraldehyde 3-phosphate dehydrogenase as an internal control to monitor equal RNA utilization.

Flow cytometry analysis

Single immune cell populations in spleens were cultured with PMA (5 ng/mL;

Sigma-Aldrich) for 6 h and then harvested for surface and intracellular staining. Additionally,

single immune cell populations in spleens were isolated from 7 days DSS-treated or 12

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vehicle-treated C57BL/6 mice. Flow cytometry data were collected with a FACS Calibur flow

cytometer (BD Biosciences, San Jose, CA). Data analyses were performed with FlowJo software

(Tree Star, Ashland, OR). The antibodies used for cell staining included anti-CD4, anti-CD8,

anti-CD19 anti-CD49b, anti-CCL5 and anti-ARC.

Protein-protein docking of ARC and TRAF6

First the three-dimensional (3-D) structures of ARC and TRAF6 were derived from the

Protein Data Bank (31) (PDB ID:1LB5 and 4UZ0). The 3-D First Fourier transform (FFT)-based protein docking algorithm of HEX 8.00 (32) was then used for docking experiments to assess the possible binding mode between ARC and TRAF6. We selected 100 sorted docked configuration possibilities for further analysis.

Dextran sulfate sodium salt (DSS) colitis and bone marrow cell transplantation mouse

models

All animal studies were approved by the University of Minnesota Institutional Animal Care

and Use Committee (IACUC). The ARC knockout (KO) mice were obtained from Albert

Einstein College of medicine of Yeshiva University. The C57BL/6 ARC wild-type (WT) mice

were purchased from JAX lab (Bar Harbor, ME).The animals were housed in climate-controlled

quarters with a 12 h light/12 h dark cycle. The mice were maintained and bred under virus- and

antigen-free conditions. 13

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For the DSS colitis model (Protocol ID: 1807-36163), C57BL/6 ARC wild-type (WT) and

ARC knockout (KO) mice (8-10 weeks old, 10 mice per vehicle group, 16 mice per DSS group,

the male female ratio was 1:1). DSS (1.5%; MW: ca 4000; Alfa Aesar, Ward Hill, MA) was added to drinking water for 7 days, then changed to fresh water for 7 days. Mice in the control

group were given normal water during the experiment. The mice were euthanized with CO2, and colon tissues were collected for further analysis.

For the bone marrow cell transplantation (Protocol ID: 1701-34463A), one week before

irradiation, the recipient mice are given acidified, antibiotic water. Water was first adjusted to pH

2.6 with concentrated hydrochloric acid and autoclaved in 1 L bottles. Then, 10 mL of 10 mg/mL neomycin in saline and 400 µL of 25 mg/mL polymyxin B sulfate in water, sterilized by

filtration, then diluted into 1 L of acidic water. Transplantation of bone marrow cells was

performed using female WT or ARC KO mice as recipients and male ARC KO mice as donors.

For isolation of bone marrow cells, male WT or ARC KO mice (10-12 weeks) were euthanized,

and their limbs were removed. Bone marrow cells were flushed from the medullary cavities of

the tibias and femurs. For the preparation of TRAF6 mutated bone marrow cells, the isolated

bone marrow cells (2 × 107 cells) were electroporated with a linearized Flag-TRAF6 (wild-type

or K201R or K461R mutant) plasmid (20 μg) at 230 V and 500 μF using the Gene Pulser X

(Bio-Rad Laboratories, Hercules, CA). Female ARC KO mice (10-12 weeks) were sub-lethally

irradiated (950 rad) using an X-ray generator and bone marrow cells (1 × 106 cells) were

tansplanted i.v. within 3 h after irradiation. At 4 weeks after irradiation, the mice were examined

by the method described previously (33). One 544 bp product representing the IL3 gene and one 14

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402 bp band representing the Y-specific Sry gene. These are the presumptive male. Only the

IL3-associated 544 bp band, which are the presumptive female. Then the successful transplanted mice were transferred to the DSS colitis model (Protocol ID: 1807-36163) for the further study.

AOM-initiated and DSS-promoted colon carcinogenesis

The AOM-initiated and DSS-promoted colon carcinogenesis model was described in our

previous study (34) and followed an approved protocol (ID: 1807-36163A). Briefly, WT and

ARC KO mice (8-10 weeks old; 6 mice per vehicle group; 24 mice per AOM/DSS group; male

female ratio was 1:1) were injected once subcutaneously with azoxymethane (AOM; Sigma, 10

mg/kg bodyweight), followed by exposure to two cycles of 3% DSS (Alfa Aesar) in drinking

water (1 cycle: 5 days of DSS, 16 days of fresh water). All animal care and experimental

procedures were performed under the guidelines of the University of Minnesota Institutional

Animal Care and Use Committee. After 50 days, the mice were euthanized with CO2, the colon

tissue was flushed with PBS. Colon lesions were measured and snap-frozen for further analysis.

The fixed colon tissue was processed by the Swiss-roll technique and embedded in paraffin for histopathological examination in a subset of animals.

Immunohistochemical Analysis

Human colon tissues were obtained from the Mayo Clinic. The baseline demographics are

shown in Supplementary Table 1. The human and mice colon tissues were supplied for immunohistochemical analysis. A Vectastain Elite ABC Kit obtained from Vector Laboratories 15

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(Burlingame, CA) was used for immunohistochemical staining according to the protocol

recommended by the manufacturer. Mice colon tissues were embedded in paraffin for

examination. Sections were stained with hematoxylin and eosin (H&E) and analyzed by

immunohistochemistry. Briefly, all specimens were deparaffinized and rehydrated. To expose antigens, samples were unmasked by submerging into boiling sodium citrate buffer (10 mM, pH

6.0) for 10 min, and then treated with 3% H2O2 for 10 min. The slide was blocked with 10% goat serum albumin in 1  PBS in a humidified chamber for 1 h at room temperature. Then, the slide

of human tissue array sections with ARC antibody (1:100) and the mouse colon tissue sections were hybridized with PCNA (1:100), CCL5 (1:100) or CXCL5 (1:100) at 4°C in a humidified

chamber overnight. The slides were washed and hybridized with the secondary antibody from

Vector Laboratories (anti-rabbit 1:150, anti-mouse 1:150 or anti-rat 1:150) for 1 h\ at room

temperature. Slides were stained using the Vectastain Elite ABC Kit (Vector Laboratories, Inc.).

After developing with 3,3’-diaminobenzidine, the sections were counterstained with hematoxylin

and observed by microscope (200) and analyzed by Image-Pro PLUS (v.6) computer software

program (Media Cybernetics, Inc.).

Statistical analysis

All quantitative data are expressed as mean values  standard deviation (S.D.) of at least

three independent experiments or samples. Significant differences were determined by a

Student’s t test or one-way ANOVA

16

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Results

Susceptibility of ARC deficient mice to DSS-induced IBD.

We established a DSS-induced experimental mouse model of IBD, in which mice were

exposed to DSS (1.5%) in drinking water for 7 days and then given normal tap water for another

7 days (Fig. 1A). We found that ARC KO mice are more susceptible to DSS exposure. ARC KO

mice exhibited more severe clinical symptoms such as loss of body weight and shortening of

colon length (Fig. 1B, C, D), indicating a greater extent of tissue damage in ARC-deficient mice.

Moreover, ARC KO mice had a more severe intestinal inflammatory response and epithelial

injury. The tissue lysates were prepared from pooled colon tissue from each mouse of each

groups. Three sets were prepared for each group and each lane shows 1 set of pooled samples

subjected to Western blotting. There is no ARC expression in ARC KO mice groups (Fig. 1E).

ARC KO mice exhibited higher expression of cytokines, especially CCL5 and CXCL5 compared

with WT mice in the DSS-induced IBD model as determined by a mouse inflammation array

(Fig. 1F). There is not markedly change in CXCL13, TNFSF8, CCL11, MPIF-2, TNFSF6,

CX3XL1, GCSF, GM-CSF, IFN-gamma, IL-1 F1, IL-1 F2, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10,

IL-12 p40/p70, IL-12 p70, IL-13, IL-17A, CXCL11, CXCL1, leptin, XCL1, CCL2, M-CSF,

CXCL19, CCL3, MIP-1 gamma, CXCL12a, TCA-3, CCL25, TIMP-1, TIMP-2, TNF-alpha,

TNFRSF1A and TNFRSF1B. Significantly higher expression of mRNA and protein levels of

CCL5 and CXCL5 were also confirmed by RT-PCR and immunohistochemical analysis, respectively (Fig. 1G, H). Notably, H&E staining showed that the colons of ARC KO mice following DSS treatment exhibited more severe inflammation in the mucosa, muscularis propria, 17

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and submucosa with the entire loss of the crypts and partial loss of the surface epithelia

compared to WT mice (Fig. 1H). Additionally, we found DSS treatment increased the expression

level of ARC in CD4-, CD19- and CD49b-positive cells. Especially, DSS treatment markedly decreased ARC expression in CD4- positive cells (Supplementary Fig. 1). These data

demonstrate a novel role for ARC in protecting against inflammation and colonic injury.

The expression of ARC is inversely correlated with CCL5 and CXCL5 levels in human

biopsy specimens.

To evaluate the role ARC in human IBD, human biopsy specimens were supplied for

further analysis. Initially, we found that ARC is expressed in both colon crypts and immune cells

in normal colon; whereas the expression level of ARC is substantially decreased in immune cells

but not in colon crypts (Fig. 2A). 40 IBD and 40 normal subjects were used. 3 different fields

were randomly selected from each sample slide to count positive immunostained and total

inflammatory cells. All fields were added together for each sample. Then average number of

cells and determined the percentage. More interestingly is that ARC is less expressed in the leukocytes (buffy coat) of IBD patients compared to normal subjects (Fig. 2B, C, D, E). There is

no different between male and female human biopsy specimens (Supplementary Fig. 2A, B).

Not surprisingly, the expression levels of CCL5 and CXCL5 are significantly higher in plasma from IBD patients (n = 40) compared with normal subjects (n = 40). Notably, ARC expression is

inversely correlated with the expression of CCL5 and CXCL5, but especially, ARC is more

closely correlated with CCL5 expression (Fig. 2F, G, H, I). 18

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ARC performs its function in CD4-positive immune cells.

Based on our findings, CCL5 is a critical cytokine associated with the function of ARC in

IBD. Thus, flow cytometry analysis was conducted to identify the immune cells in which ARC

performs its function. Our data showed that CCL5 expression is significantly increased in CD4-,

CD19-, and CD49b-positive cells, but not in CD8-positive cells, in spleens of ARC KO mice

compared with WT mice (Fig. 3A-D). Especially, CCL5 is markedly increased in CD4-positive

cells (Fig. 3A).

ARC blocks TRAF6 Lys63-linked ubiquitination regulating the TRAF6-associated NF-B signaling pathway

Tumor necrosis factor receptor-associated factors (TRAFs) have been identified as adapters controlling signaling pathways (35). TRAF molecules are essential in the regulation of

inflammation and inflammatory diseases (36,37). Initially, we found that ARC binds with

TRAF6, but not with other TRAF members in T cells (Fig. 4A). Additionally, we found ARC

does not bind with RSK2, NF-κB, IκB-α, β-catenin and EGFR (Supplementary Fig. 3). The

relationship between ARC and TRAF6 was studied by exogenously expressing these proteins in

HEK293T cells. The results showed that ARC could bind with TRAF6 (aa 288-522) (Fig. 4B, C,

D). In addition, the protein-docking model also showed that ARC could bind with TRAF6 in a

similar area (Fig. 4E). As we know, Lys63- and Lys48-linked polyubiquitination is followed by

proteasome-dependent activation and degradation, respectively (38). Intriguingly, ARC reduces 19

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TRAF6 Lys63-linked ubiquitination in exogenous systems, including in vitro ubiquitination

assay and mouse embryo fibroblasts (MEFs) of WT and ARC KO mice. ARC deficient decreased

IκB-α expression in IL-1 treated MEFs (Fig. 4F, G, H). However, knock down of ARC did not

change TRAF6 expression (Supplementary Fig. 4A), indicating that ARC did not affect

Lys48-linked ubiquitination. To identify the sites of TRAF6 that are ubiquitinated, we generated

cells expressing mutants of TRAF6 (K201R, K461R) based on mass spectrometry analysis

(Table 1) and evaluated the susceptibility of these TRAF6 mutants to ubiquitination. We

overexpressed WT-TRAF6 only, WT-TRAF6, or TRAF6 (K201R, K461R) with ARC in 293T cells. We found that ubiquitination level of TRAF6 was reduced by ARC, and this was disrupted by the TRAF6 K461 mutation but not K201 mutation (Fig. 4I). The results indicates that

ubiquitination site of K461 is important for ARC regulating TRAF6 ubiquitination. Moreover,

we also found that knocking down ARC increased the expression levels of CCL5 and CXCL5,

and also enhanced NF-B nuclear translocalization in Jurkat and MOLT3 cells (Supplementary

Fig. 4B, C, D). Overall, ARC binds with TRAF6, regulating TRAF6 Lys63-linked ubiquitination and the NF-B signaling pathway (Fig. 4J).

ARC in bone marrow-derived cells attenuates DSS-induced IBD

To address whether ARC-deficiency principally affects blood-derived immune cells,

reciprocal bone marrow transplantations were performed. Female ARC KO mice served as

recipients and were randomly assigned to groups as follows: 1) mice injected with bone marrow cells from donor male ARC KO mice; 2) mice injected with bone marrow cells from donor male 20

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WT mice; 3) mice injected with bone marrow cells with TRAF6-K201 mutation from donor

male WT mice; 4) mice injected with bone marrow cells with TRAF6-K461 mutation from donor

male WT mice; 5) mice injected with vehicle medium; 6) mice with no treatment. At 4 weeks

later, we examined the mice to confirm the success of transplantation (Supplementary Fig. 5A).

The successful transplanted mice were transferred to the DSS colitis model for further study (Fig.

5A). Results indicated that ARC KO recipients of WT cells displayed less body weight loss and

increased colon length compared with ARC KO recipients of ARC KO cells (Fig. 5B, C, D).

Additionally, bone marrow from WT donors significantly suppressed the DSS-induced increases

in CCL5 and CXCL5 levels in colon tissue (Fig. 5E, F, G and Supplementary Fig. 5B).

Importantly, WT mice exhibited reduced loss of crypts and surface epithelia compared to ARC

KO mice suffering from DSS-induced IBD. Notably, TRAF6 K461, but not the TRAF6 K201

mutation, disrupted the rescue function of WT on DSS-induced IBD in ARC KO mice (Fig. 5B,

C, D, E, F, G).

ARC-deficiency enhances colorectal tumorigenesis after AOM/DSS treatment

Colitis-associated tumorigenesis is closely associated with the duration and severity of

colonic inflammation. Earlier results (Fig. 1) demonstrated that ARC-deficient mice exhibited substantially higher susceptibility to DSS-induced IBD compared to WT mice. Here we

evaluated the protective effect of ARC in colitis-associated tumorigenesis. WT and ARC KO

mice were injected with AOM, followed by two cycles of a 7-day administration of 3% DSS

(days 7-14; 28-35), followed by normal tap water for an additional 15 days (Fig. 6A). At day 50, 21

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we found that the tumor number in ARC KO mice was significantly higher compared to WT, and

the colon length of ARC KO mice was significantly decreased compared with WT mice in the

AOM/DSS treatment groups (Fig. 6B, C, D). No significantly difference was observed between

groups in the vehicle-treated group (Fig. 6B, C, D). In addition, the ARC KO mice exhibited

significant body weight loss and had a higher mortality rate compared to WT mice (Fig. 6E, F).

The tissue lysates were prepared from pooled colon tissue from each mouse of each groups.

Three sets were prepared for each group and each lane shows 1 set of pooled samples subjected

to Western blotting. There is no ARC expression in ARC KO mice groups (Fig. 6G). ARC KO significantly enhanced the expression of mRNA level of CCL5 and CXCL5 by RT-PCR (Fig. 6H,

I). Furthermore, we found that ARC KO enhanced expression of proliferating cell nuclear

antigen (PCNA) in colon tissue compared to WT mice. The expression levels of CCL5 and

CXCL5 were significantly enhanced in colon of ARC compared to WT mice (Fig. 6J and

Supplementary Fig.6). These results confirmed that ARC suppresses colitis-associated tumor

development.

Discussion

In this study, we provide functional evidence showing that ARC mediates the inflammatory

processes in the intestine and plays a protective role in IBD and IBD-associated colorectal

tumorigenesis. Systemic deficiency of ARC exacerbated DSS-induced colitis and increased

AOM/DSS-induced tumorigenesis. We found that ARC performs this protective function in

immune cells. Notably, ARC interacts with TRAF6 and mediates ubiquitination of TRAF6 in 22

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IBD and IBD-associated tumorigenesis.

ARC is a potent inhibitor of cell death and has the unique ability to antagonize both the

intrinsic and extrinsic pathways of apoptosis (39). ARC exhibited increased expression in solid

tumors and in patients with acute myeloid leukemia and mediates the response of cells to the

induction of pharmacological apoptosis (17-20). A previous study showed that ARC is present at

high levels in most colon cancer cell lines and in almost all primary colon cancers compared with

corresponding controls, suggesting that ARC is a novel marker for human colon cancer (27).

These results indicate that ARC might be a potential target in colon cancer development.

Unexpectedly, our data revealed that ARC instead has a protective role in DSS-induced IBD and

in an AOM/DSS-induced colorectal tumorigenesis mouse model (Fig. 1, 6), but is not an

enhancer for IBD or oncogene in cancer development. The current study showed that ARC is

lower expression in leukocytes (buffy coat) of IBD patient compared with normal person (Fig.2).

There might be the link between the increased expression of ARC in primary tumors, yet decreased expression in the leukocytes (buffy coat). Further experiments should be conducted to

study the relationship. Previous reports also showed that ARC might act as a tumor suppressor in

renal cancer and myeloid tumor development (21,22). Moreover, ARC suppresses inflammatory

responses associated with inhibition of NF-κB activation (23,25,26). These interesting results led

us to uncover the mystery of ARC in the development of IBD and IBD-associated colorectal

tumorigenesis.

Our findings demonstrated that the expression level of ARC is decreased in immune cells of

IBD patients (Fig. 2A, B, E). CCL5 and CXCL5 are highly expressed in IBD patient plasma and 23

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in ARC-deficient mice in an IBD mouse model. Notably, the expression of ARC is inversely

correlated with CCL5 and CXCL5 expression (Fig. 1 and 2). CCL5 and CXCL5 are critical

cytokines, which play a crucial role in IBD (40-42). CCL5 belongs to the C-C chemokine family

and is a target gene of NF-B activity and is expressed by T lymphocytes, macrophages,

platelets, synovial fibroblasts, tubular epithelium, and certain types of tumor cells (43). CXCL5

is a member of a proangiogenic subgroup of the CXC‐type chemokine family of small, secreted

proteins. Recently, evidence showed that CXCL5 is involved in carcinogenesis and cancer

progression and is associated with NF-B activity (44,45). CCL5 is produced by T lymphocytes,

epithelial cells, fibroblasts and platelets (46). Several cell types activated by CCL5 are directly

involved in antiviral response, including NK cells (47) and CCL5 also shows

immunomodulatory functions in B cells (48).In this study, we found that ARC performs its

function by regulating CCL5 expression in T cells, B cells, and NK cells, but especially in

CD4-positive T cells (Fig. 3). These results indicate ARC should play a critical role in immune

system. ARC interacts with TRAF6, mediating the TRAF6-NF-κB signaling pathway by

regulating TRAF6 Lys63-linked ubiquitination (Fig. 4). Tumor necrosis factor

receptor-associated factors (TRAFs) were identified as adapters controlling signaling pathways

(35). The activation of TRAF6 in immune cells plays an essential role in patients with IBD (49).

The TRAF6-dependent signaling pathway leads to activation of NF-κB and mitogen-activated protein kinases (MAPKs), which are critical regulators of the immune response (50). TRAf6 K63

ubiquitination is known to activate IKK in the NF-κB signaling pathway inducing

proinflammatory cytokine expression (51). Intriguingly, our data indicated that TRAF6 Lys461 24

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could play a critical role in ARC regulation of TRAF6 Lys63-linked ubiquitination (Fig. 4I, Fig.

5). Our results suggest that ARC is key protector against IBD and IBD-associated colorectal

tumorigenesis. ARC blocks TRAF6 Lys63-linked ubiquitination and mediates the NF-κB signaling pathway.

Therapy for IBD is most often implemented in a stepwise fashion, progressing through

amino salicylates, corticosteroids, immunosuppressive medications, including ciclosporin and

tacrolimus, and finally biological therapy (52). A proportion of patients with IBD suffer an

aggressive disease course despite pharmacological treatments. Moreover, surgical intervention is

not always a viable option due to the location or extent of the disease. Fortunately, autologous

hematopoietic stem cell transplantation (HSCT) and mesenchymal stem cells (MSCs) have been

regarded as a salvage therapy for patients with severe immune-mediated diseases, including IBD

(53-56). Notably, By using ARC-deficient mice, we found that ARC in bone marrow–derived

cells could successfully attenuate colitis (Fig. 5), suggesting that ARC might be a novel

therapeutic target for IBD treatment.

In summary, our study reveals a novel role of ARC in IBD and IBD-associated colorectal tumorigenesis. We found that ARC in immune cells, especially CD4-positive T cells, interacts

with TRAF6 and regulates TRAF6 Lys63-linked ubiquitination mediating the NF-κB signaling

pathway. These data identify ARC as a potential target for IBD treatment.

Acknowledgments

The authors thank Todd Schuster for supporting experiments and Tara Adams for supporting 25

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animal experiments (The Hormel Institute, University of Minnesota). This work was supported by the Hormel Foundation (Z. Dong). This work was also supported by the Clinical Core of the

Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567).

26

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Table 1. Mass spectrometry analysis was used to identify the ubiquitination sites of ARC

1 2 3 4 5 6 Conf. Sc Prec m/z z Sequence Theor MW ∆Mass Site

QVSC*VNC*AVSMAYEEK-ubEIHD 61 8 946.6122 5 4728.1099 -0.0852 K201 § QSC*PLAN IIC*EYC*GTILIR

99 8 454.0724 6 QNHEEVMDAK-ubPELLAFQRPT 2718.3547 0.0361 K461

1, the confidence for the peptide identification; 2, the score for the peptide; 3, precursor m/z; 4, the charge for the

fragmented ion; 5, theoretical precursor molecular weight for peptide sequence; 6, the difference between theoretical

MW and experimental MW of the matching peptide sequence; *, carbamidomethyl; §, deamidated(N).

34

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Figure Legends

Figure 1. Increased susceptibility of ARC-deficient mice to DSS-induced IBD. A, WT and

ARC KO mice were administrated 1.5 % DSS for 7 days then given normal tap water for another

7 days (n = 8). Some mice were only administrated normal tap water and served as the vehicle

control group (n = 5). B, representative images of colons from WT or ARC mice in

vehicle-treated (healthy) or DSS-treated (DSS) mice are shown at day 14. C, body weight of

vehicle- and DSS-treated mice over the course of acute colitis and a percentage of original

weight. D, colon length of healthy and DSS-treated animals. E, protein levels of ARC and β-actin were detected in colon of WT and ARC KO mice. The tissue lysates were prepared from pooled

colon tissue from each mouse of each groups. Three sets were prepared for each group and each

lane shows 1 set of pooled samples subjected to Western blotting. F, cytokine expression levels in colon tissue were measured using a mouse inflammation array. Density scores were obtained using Image J software. G, mRNA expression level of CCL5 and CXCL5 was measured by

RT-PCR. H, representative H&E-stained sections of colon from vehicle- and DSS-treated mice.

CCL5 and CXCL5 expression levels were detected by immunohistochemistry (day 14). Scale bar,

100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as

mean values ± S.D. from triplicate experiments. The asterisks indicate a significant difference

between ARC KO and WT mice in vehicle- or DSS-treated groups (*, p < 0.05 and ***, p <

0.001).

35

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Figure 2. The expression of ARC in inflammatory cells is inversely correlated with CCL5

and CXCL5 expression levels in human biopsy specimens. A, H&E staining of colon from

normal and IBD tissues. Expression of ARC in normal colon or IBD patients was measured by

immunohistochemistry. Scale bar, 100 μm. B, ARC expression level in leukocytes (buffy coat) of normal or IBD biopsy specimens was detected using an immunoassay kit. Heat map across all

the samples shows the ARC levels. The level of C, CCL5 and D, CXCL5 in plasma from normal

or IBD biopsy specimens was detected using immunoassay kits. Heat map across all the samples show the CCL5 or CXCL5 levels. The analysis of E, ARC, F, CCL5, and G, CXCL5 levels in

human biopsy specimens. Based on the results from B, C, D, the correlation between ARC and H,

CCL5 or I, CXCL5 is shown. Statistical significance was determined by student t test. Data are

presented as mean values ± S.D. from triplicate experiments. The asterisks indicate a significant

difference between normal and IBD biopsy specimens (*, p < 0.05; **, p < 0.01 and ***, p <

0.001).

Figure 3. CCL5 is up-regulated in ARC KO mice. Flow cytometry analysis of the percentage

of A, CCL5/CD4-positive, B, CCL5/CD8-positive, C, CCL5/CD19-positive, or D, CCL5/CD49b cells in primary cells isolated from mouse spleen. Statistical significance was determined by

student t test. Data are presented as mean values ± S.D. from triplicate experiments. The asterisks

indicate a significant difference between WT and ARC KO specimens (*, p < 0.05 and ***, p <

0.001). 36

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Figure 4. ARC interacts with TRAF6 and blocks TRAF6 Lys63-linked ubiquitination regulating the TRAF6-associated NF-κB signaling pathway. A, ARC can bind with TRAF6 in

Jurkat and MOLT3 cells as visualized by an endogenous immunoprecipitation (IP) assay. B, to confirm the binding between TRAF6 and ARC, the Flag-TRAF6 construct was co-transfected with HA-ARC into 293T cells. After culturing for 48 h, cells were disrupted with lysis buffer and

IP with an HA antibody. The co-immunoprecipitated Flag-TRAF6 or HA-ARC was detected

using a specific antibody. C, identification of the domain of ARC that binds with TRAF6. ARC

deletion constructs were individually co-transfected with Flag-TRAF6 into 293T cells. After

culturing for 48 h, cells were disrupted with lysis buffer and immunoprecipitated with an

Flag-TRAF6 monoclonal antibody. Co-immunoprecipitated Flag-TRAF6 was detected by

Western blotting. Flag-TRAF6 or ARC was detected in whole cell lysates. D, to identify the

domain of TRAF6 binding to ARC, six Flag-TRAF6 deletion constructs were individually

co-transfected with Flag-ARC into 293T cells. After culturing for 48 h, cells were disrupted with

lysis buffer and immunoprecipitated with a Flag-ARC monoclonal antibody. The

co-immunoprecipitated Flag-ARC was detected by Western blotting. HA-TRAF6 or Flag-ARC

was detected in whole cell lysates. E, modeling of TRAF6 (blue) binding with ARC (red). F,

ARC blocks ubiquitination of TRAF6. Flag-TRAF6 was co-transfected with or without His-ARC.

The ubiquitination of TRAF6 was measured using an in vivo ubiquitination assay. The

precipitates and whole cell extracts were analyzed by Western blotting by using anti-Flag, 37

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anti-HA, or anti-His. G, in vitro, ARC deubiquitinates TRAF6. 293T cells were transfected with

Flag-TRAF6 and HA-Ub-K63. Cell lysates were immunoprecipitated with anti-Flag and then

incubated with purified His-ARC proteins at 37oC for 4 h. Ubiquitination of TRAF6 was determined by Western blotting with Flag and His antibodies. H, kinetics of TRAF6

ubiquitination. WT and ARC KO MEFs were stimulated with IL-1β for the indicated times.

Proteins from lysates were immunoprecipitated with antibody to detect TRAF6 followed by

Western blotting with an Ub-k63 or TRAF6 specific antibody to examine Ub-K63 or TRAF6

expression. Lysates were subjected to Western blotting with antibodies to detect IκBα, ARC, and

β-actin. I, WT Flag-TRAF6, mutant Flag-TRAF6 (K201R) or Flag-TRAF6 (K461R) was

co-transfected with HA-Ub-K63 and His-ARC as indicated. After immunoprecipitation with

Flag, HA-Ub-K63 or Flag-TRAF6 was detected by Western blotting. J, ARC binds with TRAF6,

regulating the TRAF6 Lys63-linked ubiquitination and NF-κB signaling pathway.

Figure 5. Bone marrow transplantation overcomes the detrimental effects of ARC

deficiency in colitis. A, generation of bone marrow chimeric mice where recipients (female)

were tail vein-injected with donor bone marrow (male, ARC KO, or WT mice) or medium as a

control. The chimeric recipients were subjected to acute colitis induction by DSS. An untreated

group served as a negative control. B, representative images of colons from ARC KO recipients in vehicle- or DSS-treated (DSS) mice are shown at day 14. C, body weight of ARC KO

recipients during the course of acute colitis and a percentage of original weight. D, colon length 38

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of ARC KO recipients. E, F, mRNA expression level of CCL5 and CXCL5 was measured by

RT-PCR. G, representative H&E-stained sections of colon from ARC KO recipients or vehicle

control. CCL5 and CXCL5 expression levels were detected by immunohistochemistry (day 14).

Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are

presented as mean values ± S.D. from triplicate experiments. The asterisks indicate a significant

difference between ARC KO and WT mice in vehicle or DSS treatment (*, p < 0.05; **, p < 0.01

and ***, p < 0.001).

Figure 6. ARC deficiency enhances IBD and IBD-induced tumorigenesis. A, WT and ARC

KO mice were injected once subcutaneously with AOM, followed by exposure to two cycles of 3%

DSS in drinking water (1 cycle: 5 days of DSS, 16 days of fresh water). B, representative images

of colon from WT or ARC mice in vehicle- or AOM/DSS-treated (AOM/DSS) mice are shown at day 50. C, the number of tumors of WT or ARC mice in vehicle- or AOM/DSS-treated

(AOM/DSS) mice. D, colon length of healthy and DSS-treated animals. E, body weight of

vehicle- and AOM/DSS-treated mice over the course of acute colitis and a percentage of original

weight. F, the survival rate of WT and ARC KO mice in healthy or AOM/DSS-treated animals. G,

protein levels of ARC and β-actin were detected in colon of WT and ARC KO mice. The tissue

lysates were prepared from pooled colon tissue from each mouse of each groups. Three sets were

prepared for each group and each lane shows 1 set of pooled samples subjected to Western

blotting. H, I, mRNA expression level of CCL5 and CXCL5 was measured by RT-PCR. J, 39

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representative H&E-stained sections of colon from vehicle- and AOM/DSS-treated mice. PCNA,

CCL5, and CXCL5 expression levels were detected by immunohistochemistry (day 50). Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean values ± S.D. from triplicate experiments. The asterisks indicate a significant difference between ARC KO and WT mice in vehicle- or DSS-treated groups (*, p < 0.05 and ***, p <

0.001.

40

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ARC is a critical protector against inflammatory bowel disease (IBD) and IBD-associated colorectal tumorigenesis

Qiushi Wang, Tianshun Zhang, Xiaoyu Chang, et al.

Cancer Res Published OnlineFirst August 19, 2020.

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