Targeting of MyD88 Homodimerization by Novel Synthetic Inhibitor TJ-M2010-5 in Preventing Colitis-Associated Colorectal Cancer
JNCI J Natl Cancer Inst (
Targeting of M yD88 Ho m odim erization by Novel Synthetic Inhibitor TJ-M 2010-5 in Preventing Colitis-Associated Colorectal Cancer
Lin Xie 0 1 2 3 4
Feng-Chao Jiang 0 1 2 3 4
Li-Min Zhang 0 1 2 3 4
Wen-Tao He 0 1 2 3 4
Jian-Hua Liu 0 1 2 3 4
Ming-Qiang Li 0 1 2 3 4
Xue Zhang 0 1 2 3 4
Shuai Xing 0 1 2 3 4
Hui Guo 0 1 2 3 4
Ping Zhou 0 1 2 3 4
Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (WTH).
0 PZ); Academy of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology , Wuhan , China ( FCJ); Department of endocrinology , Tongji
1 of Organ Transplantation, Ministry of Health, and Key Laboratory of Organ Transplantation, Ministry of Education , Wuhan, China (LX, LMZ, JHL, MQL, XZ, SX, HG
2 Affiliations of authors: Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology , Key Laboratory
3 The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions , please
4 Wuhan 430030 , China
for antitumor therapy. Background: The TLR/MyD88 signaling pathway is an important driver of inflammation and cancer and is a possible target Methods: We generated a MyD88 inhibitor (TJ-M2010-5), which was designed to bind to the TIR domain of MyD88 to interfere with its homodimerization, and the TLR/MyD88 signal pathway. We utilized a mouse model of azoxymethane/ dextran sodium sulfate (AOM/DSS)-induced colitis-associated cancer (CAC) in combination with TJ-M2010-5 administration to investigate the anti-inflammation-related cancer effect of MyD88 inhibitor in vivo. Data were analyzed with one-way and repeated measures analysis of variance. Differences in survival between groups were compared using the log rank test. All statistical tests were two-sided. Results: TJ-M2010-5 inhibited MyD88 homodimerization in transfected HEK293 cells in a concentration-dependent manner and suppressed MyD88 signaling in LPS-responsive RAW 264.7 cells in vitro. In a 10-week CAC mouse model (n = 30 per group), TJ-M2010-5 treatment statistically significantly reduced AOM/DSS-induced colitis and completely prevented CAC development with less related body mass loss, resulted in 0% mortality of treated mice (compared with 53% mortality of control mice), decreased cell proliferation, and increased apoptosis in colon tissue. TJ-M2010-5 treatment also inhibited
Novel
-
M
TJ-M
production of inflammatory cytokines and chemokines (TNαF,- IL-6,G-CSF, MIP-1β, TGF-β1, IL-11, IL-17A, IL-22 and IL-23) and
infiltration of immune cells (macrophages, dendritic cells, neutropihls an d+4CTDcells) in colon tissues of mice.
Conclusions: Our findings suggest that TLR/MyD88 signaling may be a therapeutic target for CAC intervention and MyD88
inhibitors may be a promising therapeutic modality for treating patients with colitis or CAC.
Inflammatory microenvironment around intestinal epithelial molecule myeloid differentiation factor 88 (MyD88) signaling
cells in inflammatory bowel disease plays a role in carcinog-en
pathway (
3,4
). MyD88 is one of the important adapter molecules
esis and inflammation-related epithelial cell injury and repair, of TLRs for the activation of downstream NκFB- (
5
) and
mitogenwhich contribute to the development of colitis-associated c-an
activated protein kinase (MAPK)6() pathways. Aberrant activ-a
cer (CAC) (
1,2
). Microbial infection or tissue injury can trigger
tion of TLR/MyD88 signaling is crucial for inflammation and
inflammation by activating the toll-like receptor (TLR)/adaptoris associated with the development of inflammation-related
tumorigenesis (18).
death domain (DD) and a C-terminal TIR domain separated by
a short linker region1(
9
). MyD88 can be recruited to TLR
complexes as a dimer, which is stabilized by homophilic intera-c
cancers (
7,8
) in the skin (
9,10
), ovary (11), liver (
12,13
), pancreas
(
14
), and colon (
15–17
). TLR/MyD88 signaling has been thought to
vehicle. HEK293 cells were collected 48 hours after transfection and Tumor Formation
collected for immunoprecipitation (IP) using protein A/G a-ga
The entire colon of each mouse was opened longitudinally, and
rose beads (Santa Cruz Biotech). Then, immunocomplexes ca-p
the number of tumors in each individual colon was counted.
tured by protein beads were eluted in SDS-PAGE sample buffer
With a sliding caliper, the average diameter of each tumor n-od
for western blot (WB) analysis.
ule was measured. Paraffin-embedded colon tissue sections
(4 µm) were stained by hematoxylin and eosin (H&E), and the
Kit (BD Pharmingen) according to the manufacturer’s suggested
pathological severity of colitis was evaluated by a six-grade c-las protocols. Apoptosis was detected using an In Situ Cell Death
sification system S(upplementary Method,savailable online).
Detection Fluoresce in Kit (Beyotime Biotech) with DAPI.
Immunohistochemistry, Bromodeoxyuridine, and
TUNEL (...truncated)