View
2
Download
0
Category
Preview:
Citation preview
1
A RIPK3-PGE2 circuit mediates myeloid-derived suppressor 1
cell-potentiated colorectal carcinogenesis 2
3
Guifang Yan1,2,#
, Huakan Zhao1,2,#
, Qi Zhang1,2,#
, Yu Zhou1,2
, Lei Wu1,2
, Juan Lei1,2
, Xiang Wang1,2
, 4
Jiangang Zhang1,2
, Xiao Zhang1,2
, Lu Zheng3, Guangsheng Du
4, Weidong Xiao
4, Bo Tang
5, 5
Hongming Miao6,*
, Yongsheng Li1,2,*
6
7 1Institute of Cancer,
2Clinical Medicine Research Center,
3Department of Hepatobiliary Surgery, 8
4Department of General Surgery,
5Department of Gastroenterology, Xinqiao Hospital, Third 9
Military Medical University, Chongqing 400037, China. 10 6Department of Biochemistry and Molecular Biology, Third Military Medical University, 11
Chongqing 400038, China 12
13 #
These authors contributed equally to this work. 14 *Corresponding Authors: Hongming Miao (hongmingmiao@sina.com) and Yongsheng Li 15
(yli@tmmu.edu.cn). 16
17
Running title: RIPK3-PGE2 circuit regulates MDSC. 18
19
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
2
ABSTRACT 1
Receptor-interacting protein kinase 3 (RIPK3) is essential for mucosal repair in inflammatory 2
bowel diseases (IBD) and colorectal cancer (CRC). However, its role in tumor immunity is 3
unknown. Here we report that decreased RIPK3 in CRC correlates with the accumulation of 4
myeloid-derived suppressor cells (MDSC). Deficiency of RIPK3 boosted tumorigenesis via 5
accumulation and immunosuppressive activity of MDSC. Reduction of RIPK3 in MDSC and CRC 6
cells elicited nuclear factor kappa B (NF-κB)-transcribed cyclooxygenase-2 (COX-2), which 7
catalyzed the synthesis of prostaglandin E2 (PGE2). PGE2 exacerbated the immunosuppressive 8
activity of MDSC and accelerated tumor growth. Moreover, PGE2 suppressed RIPK3 expression 9
while enhancing expression of NF-κB and COX-2 in MDSC and CRC cells. Inhibition of COX-2 10
or PGE2 receptors reversed the immunosuppressive activity of MDSC and dampened 11
tumorigenesis. Patient databases also delineated the correlation of RIPK3 and COX-2 expression 12
with CRC survival. Our findings demonstrate a novel signaling circuit by which RIPK3 and PGE2 13
regulate tumor immunity, providing potential ideas for immunotherapy against CRC. 14
15
Keywords: RIPK3; necroptosis; colorectal cancer; MDSC; COX-2; PGE2 16
17
Significance: 18
A novel signaling circuit involving RIPK3 and PGE2 enhances accumulation and 19
immunosuppressive activity of MDSC, implicating its potential as a therapeutic target in 20
anticancer immunotherapy. 21
22
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
3
Introduction 1
Colorectal cancer (CRC) is one of the most common malignant tumors and the third leading 2
cause of cancer-associated mortality in the world (1). The 5-year survival rate is only ~10% for the 3
patients with advanced CRC (2). Exploring the pathogenesis and effective therapeutic targets of 4
CRC is of great clinical significance. Inflammatory bowel diseases (IBD) are recognized as 5
precancerous diseases of CRC. The CRC-infiltrating immune cells, including myeloid-derived 6
suppressor cells (MDSC) promote the carcinogenesis, which is one of the most important causes 7
for tumor progression and therapeutic failure (3-5). MDSC can induce immune tolerance by 8
overexpressing arginase 1 (Arg-1), inducible nitric oxide synthase (iNOS or NOS2), and reactive 9
oxygen species (ROS) to suppress the activation of cytotoxic T lymphocytes (CTL). MDSC can 10
also differentiate toward tumor-associated macrophages (TAMs) and promote the 11
immunosuppressive function of regulatory T cells (Tregs). In addition, MDSC secrete 12
prostaglandin E2 (PGE2), calcium-binding protein S100A8/A9, fibroblast growth factors (FGFs), 13
matrix metalloproteinases (MMPs), transforming growth factor β (TGF-β), vascular endothelial 14
growth factor (VEGF) and other cytokines to promote tumor proliferation, angiogenesis and 15
metastasis (6). Therefore, addressing mechanisms regulating MDSC will provide new ideas for the 16
immunotherapy of CRC. 17
Inflammation initiates necroptosis which parallels with caspases-mediated apoptosis and 18
nuclear factor kappa B (NF-κB)-mediated proliferation and plays an essential role in 19
carcinogenesis (7). Receptor-interacting protein kinase 3 (RIPK3) is a central regulatory molecule 20
for necroptosis (8), whereas its role in tumor immunity remains unknown. It has been reported that 21
RIPK3 promotes the mucosal repair in IBD (9). More importantly, RIPK3 also inhibits the 22
tumorigenesis of CRC and the expression of proinflammatory factors including S100A8, 23
chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin (IL)-1β, IL-6, and tumor necrosis factor 24
alpha (TNFα) (10). Since these proinflammatory factors correlate with the accumulation and 25
maintenance of MDSC (11), the above studies suggest that RIPK3 may regulate the 26
tumor-infiltrating MDSC. 27
Here, we demonstrate that the downregulation of RIPK3 in tumor-infiltrating MDSC 28
potentiates NF-κB activation and cyclooxygenase-2 (COX-2)-derived PGE2 production. PGE2 in 29
turn further reduces RIPK3 and promotes the immunosuppressive activity of MDSC and 30
carcinogenesis. Therapy targeting this signaling circuit involving RIPK3 and PGE2 potently blunts 31
the accumulation and activity of MDSC and protects colorectal against malignancy. Our data 32
provide molecular basis for RIPK3 regulating MDSC and tumor immunity, and suggest potential 33
immunotherapeutic idea for CRC. 34
35
Methods 36
Human databases 37
The correlations between RIPK3 gene expression and CRC were determined through analysis 38
of Kaiser colon and Skrzypczak colorectal cancer datasets, respectively, which are available at 39
Oncomine (http://www.oncomine.org/). 40
The National Center for Biotechnology Information Gene Expression Omnibus databases 41
GSE21510 (12) and GSE17536 (13) containing 148 and 177 patients with CRC were evaluated for 42
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
4
the correlation of RIPK3 and indicated genes and survival, respectively. 1
2
Animal experiments 3
C57BL/6 mice were purchased from the Chinese Academy of Medical Sciences (Beijing, 4
China). RIPK3 knockout (KO) mice with C57BL/6 background were kindly provided by 5
Xiaodong Wang and Zhirong Shen (National Institute of Biological Sciences, Beijing, China). All 6
wildtype (WT) and KO mice were age and sex matched, and cages were randomly assigned to the 7
treatment groups. All animal procedures were conducted in accordance with the national and 8
international Guidelines for the Care and Use of Laboratory, approved by the Animal Care and 9
Use Committee of Third Military Medical University (Chongqing, China) and complied with the 10
Declaration of Helsinski. 11
The model of acute IBD was established by feeding C57BL/6 mice with 2% DSS dissolved 12
in sterile pure water for 6 days. For CRC induction, 6 week C57BL/6 mice were injected 13
intraperitoneally with 10mg/kg azoxymethane (AOM, Cat No. 25843-45-2, Sigma-Aldrich) and 14
after 7 days, they were fed with sterile pure water containing 2% dextran sodium sulfate (DSS, Cat 15
No. 0216011080, MW 40,000-50,000, MPbio) for 3 cycles. Colons and spleens were removed 16
upon sacrifice at indicated interval. Macroscopic tumors were measured with calipers. Portions of 17
the distal colons were either frozen in -80℃ or fixed with formaldehyde and paraffin embedded 18
for histological analysis. For some indicated experiments, GSK872 (0.75 mg/kg, Cat No. 2673, 19
Biovision), AH-6809 (5 mg/kg, Cat No. HY-10418, Med Chem Express), or ONO-AE3-208 (5 20
mg/kg, Cat No. 402473-54-5, Med Chem Express) was injected i.p. every 2 days until the mice 21
were sacrificed. Anti-Gr-1 (12.5mg/kg, Cat No. BE0075, Bioxcell) or CXCR2 antagonist 22
(CXCR2-a) SB225002 (4 mg/kg, Cat No. 182498-32-4, Med Chem Express) was injected i.p. 23
every 2 days from the third cycle until the mice were sacrificed. In the in vivo aspirin (ASA) 24
treatment experiments, mice were subjected 0.02% ASA (Cat No. 50-78-2, Med Chem Express) 25
containing water during the CRC induction. 26
Body weight, stool consistency and rectal bleeding were monitored daily. The values before 27
DSS exposure were recorded as baseline. The diarrhea scores were calculated as follows: 0-stool 28
formed pellets; 1-diarrhea; 2-hematochezia; 3-serious hematochezia or archoptosis; 4-die. The 29
average total scores were calculated in each cycle for 21 days. 30
For the chimerism experiments, C57BL/6 WT or RIPK3-KO mice were irradiated (850 cGy) 31
and injected i.v. with 10×106 bone marrow (BM) cells from congenic WT or RIPK3-KO mice, 32
respectively. For two weeks after engraftment, mice were given antibiotic water (containing 33
Trimethoprim and Sulfamethoxazole). After 7 weeks, the peripheral MDSC were analyzed to 34
confirm chimerism. 35
36
Flow cytometry (FCM) 37
The single cell suspension was prepared by mechanic dispersion and enzymatic digestion of 38
indicated tissues. For extracellular staining of target proteins, cells (1×106/ml) were preincubated 39
in a mixture of PBS, 1% fetal bovine serum (FBS), and 0.1% (w/v) sodium azide with 40
FcgIII/IIR-specific antibody to block nonspecific binding and stained with different combinations 41
of fluorochrome-coupled antibodies including CD45 (Cat No. 103108), CD11b (Cat No. 42
101208/101224), Gr-1 (Cat No. 108426), Ly6G (Cat No. 127618), Ly6C (Cat No. 128007), F4/80 43
(Cat No. 123110), CD11c (Cat No. 117308), CD206 (Cat No. 141704), CD3 (Cat No. 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
5
100206),CD8α (Cat No. 301030), CD4 (Cat No. 100422), CD33 (Cat No. 303304), HLA-DR (Cat 1
No. 307606), and APC Annexin V Apoptosis Detection Kit with 7-AAD (Cat No. 640930) from 2
Biolegend, Fixable Viability Dye EfluorTM
780 (Cat No. 65-0865-14) from eBioscience, ROS (Cat 3
No. s0033-1) from Beyotime. For intracellular staining of RIPK3 (Cat No. 95702, Cell Signaling 4
Technology; ab152130, Abcam), COX2 (Cat No. 12282, Cell Signaling Technology), Arg-1 (Cat 5
No. 42284, GeneTex), NOS2 (MA5-17139, Thermo), interferon-γ (IFN-γ, Cat No. 505810, 6
Biolegend) and granzyme B (GzmB, Cat No. 515403, Biolegend), we followed the manufacturers’ 7
protocols after cells were treated with PGE2 (Cat No. 363-24-6, Cayman Chemical), GSK872 (Cat 8
No. 2673, Biovision), AH-6809 (Cat No. HY-10418, Med Chem Express), Caffeic Acid Phenethyl 9
Ester (Cat No. S7414, Selleck), ASA (Cat No. 50-78-2, Med Chem Express), 10
N-Hydroxy-nor-L-arginine (NHNL, Cat No. 399275, Calbiochem), Bevacizumab (Bev, Roche), 11
Cetuximab (Cet, Merck), Nimotuzumab (Nim, Biotech Pharma), irinotecan (CPT-11, Pfizer), 12
Oxaliplatin (OXA, Cat No. HY-17371, Med Chem Express), 5-Fluorouracil (5-FU, Cat No. 13
HY-9006, Med Chem Express) or gemcitabine (GEM, Eli Lilly and Company), respectively. The 14
fluorescence were determined on a FACS Canto II system (BD Biosciences) and analyzed with 15
FlowJo software (Tree Star). 16
17
MDSC induction in vitro 18
MDSC were isolated and induced as indicated previously (14,15). Briefly, BM cells from WT 19
or RIPK3-/-
mice were stained by anti-mouse Gr-1 particles (Cat No.558111, BD) which were 20
optimized for positive selection and collected together using the BD IMagTM
Cell Separation 21
Magnet. Fresh harvested MDSC were incubated in RPMI1640 medium containing 5% FBS with 22
GM-CSF (20 ng/ml, 315-03, Peprotech) for 48h. 23
24
Cell culture 25
The mouse colorectal cancer CT26 cell line was purchased from ATCC (Manassas, VA) and 26
authenticated via STR profiling. Cells were cultured in RPMI-1640 (Gibco) supplemented with 10% 27
FBS (Gibco) and 1% penicillin-streptomycin (Gibco). The CT26 cells were routinely verified 28
mycoplasma-free using MycAwayTM-Color One-Step Mycoplasma Detection Kit (Yeasen 29
Bio-technol) and the most recent date of testing was April 5, 2018. Cells were used within 12 30
passages following thawing in all experiments. 31
32
CD8+ T cell isolation, purification and proliferation assay 33
CD8+ T cells were isolated from the spleen of C57BL/6 mice by CD8
+ T cell Isolation Kit 34
(Cat No. 480007, Biolegend). BM-derived MDSC were co-cultured with CFDA-SE 35
(56-carboxyfluorescein diacetatesuccinimidyl ester, CFSE; Cat No. 2011-11-2, Dojindo, 36
Kumamoro, Japan) labeled CD8+ T cells (1×10
6) at 10:1 in the medium containing anti-CD3 (1 37
μg/ml) and anti-CD28 (1 μg/ml). At day 3 post co-cultivation, cells were harvested and 38
CFSE+CD8
+ T cells were detected by FCM. 39
For the analysis of CD8+ T cell function, CD8
+ T cells were co-cultured with MDSC (5:1, 40
10:1 or 16:1) and were harvested to stimulate using Cell Stimulated Cocktail (Cat No. 4303372, 41
eBioscience) for 4 hrs and then were collected for the determination of GzmB and IFN-γ by FCM. 42
43
Western blot 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
6
Samples were lysised in RIPA buffer containing PMSF. The protein quantification was 1
determined by BCA protein assay (Cat No. P0068, Beyotime, China), and equal amounts of 2
proteins (40 μg) were subjected to SDS/PAGE (12% gels). After electrophoresis, proteins were 3
transferred onto PVDF membranes (0.45mm) in running buffer with 20% methanol. Non-specific 4
sites were blocked with 5% (w/v) non-fat dried skimmed milk powder in TBST (2M Tris-HCl 5
buffer, pH 7.6; 0.05 M NaCl; and 0.05% Tween-20) for 60 min at 37℃. The membranes were then 6
incubated overnight at 4°C with the following antibodies, which were diluted in TBST: 7
anti-RIPK3 (1:1000; Cat No. 2283, ProSci), anti-COX2 (1:500; Cat No. 12282, Cell Signaling 8
Technology), anti-p65 (1:500; Cat No. 41556, Gene Tax), anti-PKA (1:1000; Cat No. ab76238, 9
Abcam), anti-Actin (1:1000; Cat No. A1978, Sigma-Aldrich), Cell Signaling Technology 10
antibodies including anti-CREB (1:500; Cat No. 9197), anti-p-CREB (1:500; Cat No. 9198), 11
anti-p-stat3 (1:1000; Cat No. 9145), anti-stat3 (1:1000; Cat No. 4904), anti-p-stat6 (1:1000; Cat 12
No. 56554s), and anti-stat6 (1:1000; Cat No. 5397). After four washes in TBST, the membranes 13
were incubated with horseradish-peroxidase conjugated secondary antibodies (Cat No. A0562, 14
Beyotime) for 1 h in TBST (dilution of 1:5000). Protein bands were visualized by using Enhanced 15
Chemiluminescence (ECL) Plus Western blotting detection kit (Cat No. P0018-2, Beyotime). 16
17
Confocal microscopy. 18
Mice or human tissues were fixed and permeabilized with Fixation & Permeabilization 19
Buffers (BD Biosciences) for 15min and then incubated with FC-block (BD Biosciences) for 30 20
min at room temperature. Subsequently, cells were stained with Gr-1 (1:50, Cat No. MAB1037, 21
R&D Systems), RIPK3 (1:100, Cat No. 95702, Cell Signaling Technology), or CD33 (1:100, Cat 22
No. ab213050, Abcam), RIPK3 (1:50, Cat No. ab152130, Abcam) for overnight at 4 °C, washed 23
thrice with PBS before incubation with fluorochrome associated secondary antibodies for 30min 24
with Alexa-488, and 647 (Bioss). Afterwards, sections were washed thrice with wash buffer (BD 25
Biosciences), and then were incubated with DAPI and mounted on slides using Prolong Gold 26
antifade reagent (Beyotime). The sections were imaged with a Leica TCS SP5 laser scanning 27
confocal microscope (Leica Microsystems). The co-localization and average intensity were 28
assessed by using Leica LASX (Microsystems software). 29
30
Immunohistochemistry analysis 31
Colon and tumor tissues were fixed with formaldehyde. Paraffin sections were stained with 32
hematoxylin and eosin or subjected to immunohistochemistry for Gr-1 (Cat No. MAB1037, R&D 33
Systems), RIPK3 (Cat No. 2283, ProSci) and COX-2 (Cat No. 12282, Cell Signaling Technology). 34
35
Real-time PCR 36
Real-time PCR was performed as previously (16). Total RNA was extracted from cells with 37
RNA queousTM
Mico kit (Cat No. 00490515, Invitrogen). Real-time quantitative PCR was 38
performed on a CFX384TM
system (BIO-RAD). Primers used in this study are below: RIPK3-F: 39
CAG TGG GAC TTC GTG TCC G, RIPK3-R: CAA GCT GTG TAG GTA GCA CAT C; EP1-F: 40
CTT AAC CTG AGC CTA GCG GAT, EP1-R: ATG TGC CAT TAT CGC CTG TTG; EP2-F: 41
GGA GGA CTG CAA GAG TCG TC, EP2-R: GCG ATG AGA TTC CCC AGA ACC; EP3-F: 42
GCT CAT GGG GAT CAT GTG TGT, EP3-R: CAC CAC CCC GAA GAT GAA CAT; EP4-F: 43
ACC ATT CCT AGA TCG AAC CGT, EP4-R: CAC CAC CCC GAA GAT GAA CAT; ACTIN-F: 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
7
TGA CAG GAT GCA GAA GGA GA; ACTIN-R: GTA CTT GCG CTC AGG AGG AG. 1
2
PGE2 determination 3
Cell supernatants were collected for evaluating PGE2 concentration with UPLC-MS/MS as 4
previously (16). Prior to sample extraction, d4-PGE2 (500 pg) was added to permit quantification. 5
Extracted samples were separated by an Acquity UPLC I-Class system (Waters, MA, USA) and 6
mass spectrometry was performed on an AB Sciex 6500 QTRAP. PGE2 was analyzed using 7
scheduled multiple reaction monitoring (MRM). Data acquisitions were performed using Analyst 8
1.6.2 software (Applied Biosystems). 9
10
Cell proliferation assay 11
Cell counting kit-8 (CCK8) assay was used to assess the proliferation of MDSC and CT-26 12
cells. For indicated experiments 5 × 105 BM-derived MDSC or 5 × 10
3 CT-26 cells were seeded in 13
96-well plates. After 48 hrs, a batch of cells in 100 μl medium was stained with 10 μl of CCK8 14
reagent (Dojindo, Kumamoto, Japan) at 37℃ for 2 hrs. The data was quantified with an automatic 15
plate reader (Thermo) at 450 nm. 16
17
Statistics 18
The number of animals used in the experiments was estimated to give sufficient power 19
(>90%) on the basis of the effect sizes observed in our preliminary data. The statistical analysis 20
was performed using Excel (Microsoft), Origin 9.1 (OriginLab) or GraphPad Prism 7 (GraphPad 21
Software). Statistical significance for binary comparisons was assessed by 2-tailed Student’s t test. 22
For comparison of more than 2 groups, ANOVA with Sidak’s multiple comparisons test was used. 23
For correlation analysis, Pearson’s correlation coefficient was applied. Overall survival was 24
calculated using the Kaplan-Meier method, and the differences in survival curves were analyzed 25
using the log-rank test. All data are reported as mean ± SEM. The P value of 0.05 or less was 26
considered significant. 27
28
Results 29
RIPK3 is downregulated in CRC-infiltrating MDSC 30
The RIPK3 expression was first evaluated in CRC patient databases with Oncomine which 31
showed a consistent decrease of RIPK3 in CRC tissue (Supplementary Fig. S1A). We next 32
employed AOM plus DSS-induced mouse CRC model (Fig. 1A, left panel) (17). The body weight 33
reduced during the DSS treatment and rebound subsequently after DSS withdrawn at each cycle 34
(Supplementary Fig. S1B). Upon sacrifice after Day 90, the colorectal was collected and tumors 35
were separated (Fig. 1A, right panel). We compared the percentage of immune cells in tumor and 36
colorectal tissue and found that both leukocytes (CD45+) and MDSC (CD11b
+Gr-1
+) were 37
significantly higher in tumor than in colorectal tissues (Fig. 1B and 1C). The tumor-infiltrating 38
MDSC showed lower RIPK3, compared with MDSC in the colorectal of tumor-bearing mice (Fig. 39
1D and 1E). 40
We collected clinical colorectal cancer and adjacent normal tissues and found that the 41
accumulation of MDSC was much higher. Consistently, RIPK3 expression in MDSC was 42
significantly suppressed in the tumor microenvironment (TME) than in adjacent tissue (Fig.1F). 43
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
8
Of interest, we found that RIPK3 expression was highest in the colorectal of IBD mice whereas 1
was lower in the colorectal of CRC mice and lowest in tumor tissues (Supplementary Fig. 2
S1C-S1E), suggesting a differential RIPK3 expression pattern during the development of CRC. In 3
addition, we evaluated other immune cells in colorectal and tumor tissue of CRC mice. CD8+ T 4
cells and dendritic cells (DCs, CD11c+) were less while macrophages (MΦs, F4/80
+) were more 5
abundant in tumor than in colorectal tissues (Supplementary Fig. S1F). We also determined RIPK3 6
expression but did not found significant changes in DCs, MΦs, or T cells when compared to that 7
in colorectal tissues (Supplementary Fig. S1G). We hence wondered whether the down-regulation 8
of RIPK3 in tumor-infiltrating MDSC was caused by factors from TME. We stimulated mouse 9
BM cells with supernatants from CT26 colorectal cancer cells in vitro and found that the 10
percentage of MDSC increased significantly whereas the expression of RIPK3 was 11
down-regulated significantly (Fig.1G). Together these results indicated that RIPK3 was 12
downregulated in tumor tissues and CRC-infiltrating MDSC. 13
14
Enhanced MDSC accumulation and tumorigenesis in RIPK3 deficient mice 15
To investigate the role of RIPK3 in the tumorigenesis of CRC, we employed RIPK3 16
knockout mice (KO). These mice showed decreased body weight, higher diarrhea score, shortened 17
colorectal length, increased tumor number in colorectal, heavier spleen, and significantly reduced 18
survival, compared with wildtype (WT) mice (Fig. 2A-2E; Supplementary Fig. S2A). The 19
accumulation of MDSC in tumor, colorectal and spleen also increased in KO mice (Fig. 2F-2I). Of 20
note, only the granulocytic MDSC (g-MDSC, CD11b+Ly6G
+) increased in the tumor compared 21
with that in the colorectal tissues, which was not shared by the monocytic MDSC (m-MDSC, 22
CD11b+Ly6C
+) (18), MΦs, DCs, or T cells (Fig. 2J and 2K). 23
We also used GSK872, a specific RIPK3 inhibitor (19) to treat the WT mice. GSK872 24
significantly aggregated AOM plus DSS induced weight loss, colorectal shortening, tumor mass, 25
splenomegaly and MDSC accumulation, while it did not alter the infiltration of MΦs and DCs 26
(Supplementary Fig. S2B-S2H). These results demonstrated that RIPK3 deficiency promoted 27
colorectal carcinogenesis and MDSC infiltration. 28
29
Deficiency of RIPK3 promotes the proliferation and immunosuppressive activity of MDSC 30
in vitro 31
The role of RIPK3 on MDSC was next sought in vitro. We found that the percentage of 32
MDSC was much higher in RIPK3-KO group than in WT after mouse BM cells were stimulated 33
by GM-CSF (Fig. 3A), although there was no difference between WT and RIPK3-KO groups 34
without stimulation (Supplementary Fig. S3A). RIPK3 absence in MDSC also resulted in a 35
modest higher proliferation (Fig. 3B) but did not show significant change in cell death (Fig. 3C), 36
which were consistent with the results from GSK872-treated MDSC (Supplementary Fig. S3B). 37
The differentiation of MDSC was also assessed in vitro. After induction with GM-CSF (20 ng/ml) 38
for 48 hrs, the percentage of MΦs, especially M2 type MΦs (F4/80+CD206
+) were significantly 39
higher in KO group, while DCs were lower as compared with WT (Fig. 3D), suggesting that 40
RIPK3 absence in MDSC promoted the M2-like differentiation upon GM-CSF induction. 41
In addition, Arg-1 but not NOS2 or ROS increased in the RIPK3-KO and GSK872-treated 42
MDSC (Fig. 3E and 3F; Supplementary Fig. S3C). The co-cultivation of RIPK3-KO and 43
GSK872-treated MDSC significantly dampened anti-CD3 and anti-CD28 induced proliferation of 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
9
CD8+ T cells (Fig. 3G), as well as the expression of GzmB and IFN-γ (Fig. 3H; Supplementary 1
Fig. S3D), compared with that co-cultured with WT MDSC. Administration of NHNL, an Arg-1 2
inhibitor, significantly rescued the activity of CD8+ T cells (Fig. 3I). Of note, GSK872-treated T 3
cells also showed moderately impaired expression of GzmB and IFN-γ (Supplementary Fig. S3E), 4
suggesting that RIPK3 deficiency in CTL may also contribute to colorectal carcinogenesis. 5
Furthermore, the supernatant from RIPK3-KO MDSC enhanced the proliferation of CT26 cells 6
(Fig. 3J). These results indicated that RIPK3 deficiency enhanced proliferation and 7
immunosuppressive function of MDSC. 8
9
Carcinogenesis is accelerated after RIPK3-deficient BM chimerism 10
To further test the role of RIPK3 on MDSC function in vivo, we generated chimeras by 11
infusing WT or KO BM cells into WT or KO recipient mice after irradiation. The presence of 12
chimerism after 7 weeks was confirmed using FCM. These animals were subsequently induced 13
CRC using model 2 (Supplementary Fig. S3F). We found that the WT recipients exhibited more 14
severe weight loss, higher mortality and tumor formation ratio, shorter colorectal length, 15
splenomegaly and more MDSC in colorectal and spleen when engrafted with cells from 16
RIPK3-KO donors, compared with that from WT donors (Fig. 4A-4G). The percentage of 17
g-MDSC were higher, while MΦs and DCs showed no significant difference in the colorectal and 18
spleen of mice received KO BM, compared with that of mice received WT BM. The leukocytes in 19
colorectal tissues were higher in mice received KO BM, while their percentages did not show 20
significant change in spleen (Fig. 4H and 4I; Supplementary Fig. S3G and S3H). Moreover, 21
although KO recipients showed a moderate weight loss, increased mortality and modest higher 22
tumorigenecity compared with WT recipients after engrafted with WT BM cells, the colorectal 23
length, spleen weight, MDSC infiltration, MΦs and DCs in colorectal and spleen did not show 24
significant change (Fig. 4A-4I). 25
To further validate the essential role of MDSC in the tumorigenesis of CRC, we 26
administrated anti-Gr-1 (to deplete MDSC) and CXCR2-a SB225002 (to inhibit MDSC 27
chemotaxis) every 2 days from the third cycle of DSS until the mice were sacrificed. Both 28
anti-Gr-1 and CXCR2-a reversed the weight loss, mortality, tumor formation, colorectal length 29
and the MDSC infiltration in colorectal and spleen compared with KO control (Fig. 4J-4O). 30
Together these findings supported the conclusion that RIPK3 deficiency in MDSC promoted 31
tumorigenesis. 32
33
NF-κB/COX-2/PGE2 axis is upregulated in RIPK3-deficient MDSC 34
We next explored the underlying mechanism by which RIPK3 regulated MDSC. 35
Aforementioned, PGE2 is a pro-inflammatory and immunosuppressive lipid mediator that 36
potentiates MDSC activity and tumor growth (20). COX-2 is an essential enzyme for the 37
production of PGE2. We found that COX-2 expression was significantly upregulated in the 38
tumor-infiltrating MDSC than in colorectal MDSC (Fig. 5A), but no significant difference of 39
COX-2 expression was observed in CD45- cells of tumor and colorectal tissues (Supplementary 40
Fig. S4A). Compared with WT mice, COX-2 expression were upregulated in tumor-infiltrating 41
MDSC (Fig. 5B) and in CD45- cells of both tumor and colorectal tissues of RIPK3-KO mice 42
(Supplementary Fig. S4B and S4C). Using UPLC-MS/MS, we found that RIPK3-KO MDSC 43
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
10
produced more PGE2 than that in WT MDSC (Fig. 5C), which was consistent with the results of 1
COX-2 expression. 2
Given that NF-κB is a well-known transcription factor of COX-2 and an essential controller 3
for the immunosuppressive activity of MDSC (21,22), we examined the expression of NF-κB in 4
MDSC. Significant upregulated NF-κB p65 and COX-2 were observed in RIPK3-KO MDSC, 5
compared with WT (Fig. 5D). We administrated aspirin (ASA, COX inhibitor) and caffeic acid 6
phenethyl ester (CAPE, NF-κB inhibitor) (23) and found that they both inhibited PGE2 production 7
from RIPK3-KO MDSC but only showed a trend in decreasing PGE2 production from 8
GSK-872-treated CT26 cells (Supplementary Fig. S4D and S4E). We also assessed other key 9
signaling molecules that drive the accumulation and function of MDSC including stat3 and stat6. 10
However, they showed no difference between WT and KO MDSC (Supplementary Fig. S4F). 11
These findings demonstrated that RIPK3 reduction in MDSC promoted the activation of 12
NF-κB/COX-2/PGE2 axis. 13
14
Inhibitors targeting COX-2 and EP2 blunt the immunosuppressive activity of MDSC and 15
carcinogenesis 16
PGE2 exerts its function by binding to its receptors including EP1-4. We found that the 17
RIPK3-KO MDSC showed higher EP2 and EP4, compared with WT MDSC (Supplementary Fig. 18
S5A). Therefore, the CRC mice model was treated with ASA or EP inhibitors (EP1 and EP2 19
inhibitor AH6809 and EP4 inhibitor ONO-AE3-208) (24). We found that ASA significantly 20
protected the mice against tumorigenesis and reduced the accumulation and COX-2/Arg-1 21
expression of MDSC (Fig. 6, A-E; Supplementary Fig. S5B). AH6809 but not ONO-AE3-208 22
attenuated AOM plus DSS induced tumorigenesis and MDSC accumulation (Fig. 6F-6L; 23
Supplementary Fig. S5C). 24
In vitro, PGE2 significantly enhanced Arg-1 expression in MDSC and the differentiation 25
toward M2 macrophages, which were reversed by ASA and AH6809 (Fig. 6M and 6N). 26
Antagonists of NF-κB/COX-2/PGE2/EPs signaling pathway consistently rescued the CD8+ T cell 27
activation dampened by RIPK3-KO MDSC co-cultivation (Fig. 6O and 6P). 28
In addition, PGE2 promoted the proliferation of CT26 cells, which was blunted by AH6809 29
(Supplementary Fig. S5D, left panel). The co-cultivation with supernatant from RIPK3-KO 30
MDSC also enhanced the proliferation of CT26 cells which was reversed by ASA or AH6809 31
pretreatment in these MDSC (Supplementary Fig. S5D, right panel). Together these results 32
indicated that antagonists targeting NF-κB/COX-2/PGE2 signaling improved the prognosis of 33
CRC. 34
35
RIPK3-PGE2 circuit in tumor microenvironment potentiates malignancy 36
We next questioned whether PGE2 in turn regulated RIPK3 and the downstream 37
NF-κB/COX-2 signaling. BM-derived MDSC were treated with or without PGE2, and we indeed 38
found that PGE2 significantly suppressed RIPK3 while enhanced the expression of p65 and 39
COX-2 in MDSC (Fig. 7A). 40
It is well-known that EP receptors are G-protein coupled receptors that activate cAMP 41
dependent protein kinase A (PKA) and promote the subsequent translocation of the transcription 42
factor cAMP responsive element binding protein (CREB) (25). Of note, a recent report indicated 43
that CREB reduced the promoter activity of RIPK3 (26). Here we found that the 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
11
PGE2-downregulated RIPK3 could be rescued by both H89 (PKA inhibitor) and AH6809 in 1
MDSC (Fig. 7B and 7C). ASA also could upregulate RIPK3 expression in MDSC (Fig. 7D), 2
indicating that PGE2 suppressed RIPK3 via PKA-CREB signaling. Consistently, PGE2 decreased 3
RIPK3 via PKA-CREB pathway while both PGE2 and GSK872 promoted the expression of p65 4
and COX-2 in CT26 cells (Supplementary Fig. S6A-S6D). These results identified a novel circuit 5
involving RIPK3, NF-κB, COX-2, and PGE2 in the tumor microenvironment. 6
To explore the correlation of clinical chemotherapy and targeted therapy with RIPK3 7
expression, we treated MDSC with some common drugs used in cancer. The expression of 8
RIPK3 in MDSC were upregulated by bevacizumab or cetuximab (Fig. 7E) but was 9
downregulated by CPT-11, OXA, 5-FU and GEM (Fig. 7F). 10
We also evaluated the clinical relevance of the RIPK3-PGE2 circuit in CRC cancer patients. 11
We found that with the development of CRC clinical stage, the expression of RIPK3 in 12
tumor-infiltrating MDSC decreased (Fig. 7G; Supplementary Table 1). The relationship between 13
RIPK3 and indicated gene transcripts were examined in 148 patients with CRC from National 14
Center for Biotechnology Information Gene Expression Omnibus database (GSE21510) (13). Our 15
results demonstrated that RIPK3 expression negatively correlated with CD33 and S100A8 which 16
are MDSC markers (27) (Fig. 7H). Importantly, RIPK3 also negatively correlated with PTGS2 17
(COX-2) (Fig. 7I). Since the database GSE21510 lacked the survival results, we analyzed the 18
correlation of RIPK3 and survival of CRC patients with another one (GSE17536) which involved 19
177 patients. We divided patients into “low” and “high” groups based on the median values of 20
RIPK3 and PTGS2. We found that RIPK3high
PTGS2low
patients showed longest survival while low 21
RIPK3 and high PTGS2
was associated with poor survival (Fig. 7J; Supplementary Table 2). 22
Therefore RIPK3 downregulation and COX-2/PGE2 upregulation in the tumor microenvironment 23
formed a circuit that promoted the accumulation of immunosuppressive MDSC and colorectal 24
carcinogenesis. 25
26
DISCUSSION 27
The infiltration of MDSC in tumor microenvironment is closely related to poor prognosis 28
(3,11). Here, we found that that the down-regulation of RIPK3 promoted the infiltration and 29
immunosuppressive activity of MDSC in tumor microenvironment. The chimeric mice experiment 30
also indicated the pivotal role of RIPK3 on CRC-infiltrating MDSC. Therefore, we identified that 31
RIPK3 regulated tumor immunity by modulating MDSC. 32
MDSC play an immunosuppressive function mainly via multiple signal pathways. First, the 33
lipid metabolite PGE2 derived from arachidonic acid via COX-2 catalysis in tumor 34
microenvironment stimulates the expression of Arg-1, IL-6, VEGF and other cancer-promoting 35
molecules in MDSC (11,28). Of note, MDSC also express COX-2 which promotes their own 36
immunosuppressive activity (29). Second, NF-κB activation in MDSC can promote the 37
proliferation and inhibit the differentiation of MDSC (22). Third, the secretion of VEGF from 38
MDSC is also promoted by stat signaling pathway, which enhances angiogenesis (11). Our present 39
study showed that the expression of COX-2 in RIPK3-KO MDSC of CRC tissues was 40
significantly enhanced, compared with that in WT MDSC. In vitro experiments also demonstrated 41
that RIPK3 deficient MDSC exhibited increased COX-2 expression and PGE2 secretion. However, 42
we did not observe significant changes in ROS, NOS2, stat3 and stat6 between WT and 43
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
12
RIPK3-KO MDSC. These data suggested that the loss of RIPK3 in tumor-infiltrating MDSC 1
promoted the immunosuppressive function by activating COX-2/PGE2. 2
A previous study reported a necrosis-independent pathway of IBD by regulating DCs (9). 3
They showed that in DSS-induced IBD, RIPK3 deficiency impaired NF-κB activation and caspase 4
1-mediated processing of IL-1β in DCs, thereby dampened the tissue repair. Actually, we also 5
observed that RIPK3 was upregulated in the colorectal tissue during the IBD acute induction of 6
DSS, while it significantly reduced at the stage of CRC. Furthermore, we showed that the 7
RIPK3-KO MDSC did not tend to differentiate into DCs. These cells possessed higher 8
immunosuppressive function and were prone to differentiate toward M2 macrophages. Another 9
recent report showed that RIPK3 deficiency enhanced lipopolysaccharide (LPS) induced IL-1β 10
and TNFα expression in macrophages (30). Since LPS is an endotoxin that was found in the outer 11
membrane of Gram-negative bacteria such as E.coli (31) and NF-κB also transcripts IL-1β and 12
TNFα (32), this study was consistent with our results that RIPK3 deficiency in MDSC enhanced 13
NF-κB activation that in turn upregulated COX-2 expression and PGE2 production during the 14
carcinogenesis of CRC. 15
The mechanisms of RIPK3 upregulating NF-κB are complex, which we did not investigate in 16
detail in this study but discuss below. Aforementioned NF-κB is a parallel proliferation pathway of 17
RIPK3-mediated necroptosis and caspases-associated apoptosis (7,33). Knockout of RIPK3 is 18
supposed to lead to a compensatory promotion of NF-κB pathway. A recent study indicated that 19
RIPK3 absent activated NF-κB via a MLKL-independent pathway (34). Moreover, ubiquitination 20
degradation is an important mechanism for the down-regulation of multiple transcription factors in 21
cells. It has been shown that Cullin-RING E3 ligases (CRLs) can mediate NF-κB ubiquitination 22
degradation and reduce its entry into nuclei (35). Hence RIPK3 may also phosphorylate CRLs by 23
mimicking MLKL activation, thereby promoting ubiquitination degradation of NF-κB. 24
Of note, the downregulation of RIPK3 and the subsequent NF-κB/COX-2/PGE2 signaling 25
was also identical in CRC cells in vivo and in vitro, which demonstrated the negative correlation 26
between RIPK3 expression and tumorigenesis. Administration of PGE2 inhibited RIPK3 27
expression but enhanced NF-κB/COX-2 signaling in both MDSC and CT26 colorectal cancer cells 28
via activating PKA-CREB signaling, indicating an unappreciated negative signaling circuit that 29
aggregate the malignancy. Moreover, PGE2 was reported to directly and indirectly blunt the 30
activation of CD8+ T cells (20,36). Our results showed that inhibition of COX-2 or the PGE2 31
receptors significantly reversed the downregulated RIPK3 and attenuated the immunosuppressive 32
activity of MDSC thereby dampened the tumorigenesis of CRC. 33
In summation, our present study identified a novel RIPK3-PGE2 circuit that regulated the 34
infiltration and function of MDSC and the tumorigenesis of CRC. RIPK3 reduction led to NF-κB 35
activation and upregulation of downstream COX-2 which catalyzed the synthesis of PGE2. PGE2 36
in turn further inhibited RIPK3 and promoted NF-κB/COX-2 and Arg-1 expression in MDSC. 37
This signaling circuit also existed in CRC cells and accelerated tumor growth. Importantly, using 38
compounds or drugs targeting this signaling circuit clarified the immunoregulatory role of RIPK3 39
and attenuated the carcinogenesis of CRC. Daily consumption of low-dose ASA has now been 40
applied to efficiently prevent and cure CRC (37,38), delineating the significance of PGE2 41
blockade in the tumor microenvironment. PGE2 was reported to directly stimulate CRC cells to 42
secrete CXCL1 which bound to CXCR2 to recruit MDSC into TME (39,40). Of note, CRC cells 43
also express CXCR2 which predicts poor prognosis. The CXCR2 antagonist inhibits the 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
13
proliferation and metastasis of CRC cells (41,42). Since the RIPK3-PGE2 circuit exists in both 1
MDSC and CRC cells, our results demonstrated a mutual role of PGE2 blockade and CXCR2 2
antagonist in inhibiting CRC tumorigenesis in RIPK3-KO mice. Moreover, our data demonstrated 3
that cetuximab and bevacizumab upregulated RIPK3, while CPT-11, OXA, 5-FU and GEM 4
suppressed RIPK3 in MDSC. Of interest, cetuximab or bevacizumab are both the first-line 5
targeted drugs for CRC patients and they were reported to inhibit COX2 (43,44). Therefore, 6
targeting RIPK3 in MDSC might be considered for rational use of chemotherapeutic and targeted 7
drugs, which is essential for re-educating the immunosuppressive TME and enhance the 8
anti-tumor immunity. These findings provided molecular basis and potential ideas for the 9
immunotherapy of CRC. 10
11
Disclosure of Potential Conflicts of Interest 12
The authors have declared that no conflict of interest exists. 13
14
Grant Support 15
The work was supported by Youth 1000 Talent Plan (to Y. Li) and the National Natural 16
Science Foundation of China (81472435 and 81671573 to Y. Li) and cstc2017jcyjBX0071 (to H. 17
Miao) from the Foundation and Frontier Research Project of Chongqing. 18
19
Acknowledgments 20
We sincerely thank Xiaodong Wang and Zhirong Shen (National Institute of Biological 21
Sciences, Beijing, China) for providing RIPK3 knockout (KO) mice. We extremely appreciate 22
Chunyan Hu for her support on FCM. We are also very grateful to Rong Xin and Lu Jiang for their 23
valuable assistance in IHC and IF procedure, and to Jin Peng and Qian Chen for confocal 24
microscopy experiments. 25
26
References 27
1. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin 2017;67:7-30. 28
2. Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet 2014;383:1490-502. 29
3. Medina-Echeverz J, Aranda F, Berraondo P. Myeloid-derived cells are key targets of tumor 30
immunotherapy. Oncoimmunology 2014;3:e28398. 31
4. Katoh H, Wang D, Daikoku T, Sun H, Dey SK, Dubois RN. CXCR2-expressing 32
myeloid-derived suppressor cells are essential to promote colitis-associated tumorigenesis. 33
Cancer Cell 2013;24:631-44. 34
5. Tauriello DVF, Batlle E. Targeting the Microenvironment in Advanced Colorectal Cancer. 35
Trends Cancer 2016;2:495-504. 36
6. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune 37
system. Nat Rev Immunol 2009;9:162-74. 38
7. Han J, Zhong CQ, Zhang DW. Programmed necrosis: backup to and competitor with apoptosis 39
in the immune system. Nat Immunol 2011;12:1143-9. 40
8. Weinlich R, Oberst A, Beere HM, Green DR. Necroptosis in development, inflammation and 41
disease. Nat Rev Mol Cell Biol 2017;18:127-36. 42
9. Moriwaki K, Balaji S, McQuade T, Malhotra N, Kang J, Chan FK. The necroptosis adaptor 43
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
14
RIPK3 promotes injury-induced cytokine expression and tissue repair. Immunity 1
2014;41:567-78. 2
10. Bozec D, Iuga AC, Roda G, Dahan S, Yeretssian G. Critical function of the necroptosis 3
adaptor RIPK3 in protecting from intestinal tumorigenesis. Oncotarget 2016;7:46384-400. 4
11. Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: linking inflammation and 5
cancer. J Immunol 2009;182:4499-506. 6
12. Tsukamoto S, Ishikawa T, Iida S, Ishiguro M, Mogushi K, Mizushima H, et al. Clinical 7
significance of osteoprotegerin expression in human colorectal cancer. Clin Cancer Res 8
2011;17:2444-50. 9
13. Smith JJ, Deane NG, Wu F, Merchant NB, Zhang B, Jiang A, et al. Experimentally derived 10
metastasis gene expression profile predicts recurrence and death in patients with colon cancer. 11
Gastroenterology 2010;138:958-68. 12
14. Lechner MG, Liebertz DJ, Epstein AL. Characterization of cytokine-induced myeloid-derived 13
suppressor cells from normal human peripheral blood mononuclear cells. J Immunol 14
2010;185:2273-84. 15
15. Dolcetti L, Peranzoni E, Ugel S, Marigo I, Fernandez Gomez A, Mesa C, et al. Hierarchy of 16
immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by 17
GM-CSF. Eur J Immunol 2010;40:22-35. 18
16. Li Y, Dalli J, Chiang N, Baron RM, Quintana C, Serhan CN. Plasticity of leukocytic exudates 19
in resolving acute inflammation is regulated by MicroRNA and proresolving mediators. 20
Immunity 2013;39:885-98. 21
17. Neufert C, Becker C, Neurath MF. An inducible mouse model of colon carcinogenesis for the 22
analysis of sporadic and inflammation-driven tumor progression. Nat Protoc 23
2007;2:1998-2004. 24
18. Ouzounova M, Lee E, Piranlioglu R, El Andaloussi A, Kolhe R, Demirci MF, et al. Monocytic 25
and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal 26
tumour plasticity during metastatic cascade. Nat Commun 2017;8:14979. 27
19. Qiu X, Klausen C, Cheng JC, Leung PC. CD40 ligand induces RIP1-dependent, 28
necroptosis-like cell death in low-grade serous but not serous borderline ovarian tumor cells. 29
Cell Death Dis 2015;6:e1864. 30
20. Zelenay S, van der Veen AG, Bottcher JP, Snelgrove KJ, Rogers N, Acton SE, et al. 31
Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity. Cell 32
2015;162:1257-70. 33
21. Nguyen LK, Cavadas MA, Kholodenko BN, Frank TD, Cheong A. Species differential 34
regulation of COX2 can be described by an NFkappaB-dependent logic AND gate. Cell Mol 35
Life Sci 2015;72:2431-43. 36
22. Yu J, Wang Y, Yan F, Zhang P, Li H, Zhao H, et al. Noncanonical NF-kappaB activation 37
mediates STAT3-stimulated IDO upregulation in myeloid-derived suppressor cells in breast 38
cancer. J Immunol 2014;193:2574-86. 39
23. Natarajan K, Singh S, Burke TR, Jr., Grunberger D, Aggarwal BB. Caffeic acid phenethyl 40
ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. 41
Proc Natl Acad Sci U S A 1996;93:9090-5. 42
24. Rosch S, Ramer R, Brune K, Hinz B. Prostaglandin E2 induces cyclooxygenase-2 expression 43
in human non-pigmented ciliary epithelial cells through activation of p38 and p42/44 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
15
mitogen-activated protein kinases. Biochem Biophys Res Commun 2005;338:1171-8. 1
25. Alvarez Y, Municio C, Alonso S, Sanchez Crespo M, Fernandez N. The induction of IL-10 by 2
zymosan in dendritic cells depends on CREB activation by the coactivators CREB-binding 3
protein and TORC2 and autocrine PGE2. J Immunol 2009;183:1471-9. 4
26. Guida N, Laudati G, Serani A, Mascolo L, Molinaro P, Montuori P, et al. The neurotoxicant 5
PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 6
and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death. 7
Biochem Pharmacol 2017;142:229-41. 8
27. Heinemann AS, Pirr S, Fehlhaber B, Mellinger L, Burgmann J, Busse M, et al. In neonates 9
S100A8/S100A9 alarmins prevent the expansion of a specific inflammatory monocyte 10
population promoting septic shock. FASEB J 2017;31:1153-64. 11
28. Condamine T, Gabrilovich DI. Molecular mechanisms regulating myeloid-derived suppressor 12
cell differentiation and function. Trends Immunol 2011;32:19-25. 13
29. Rodriguez PC, Hernandez CP, Quiceno D, Dubinett SM, Zabaleta J, Ochoa JB, et al. Arginase 14
I in myeloid suppressor cells is induced by COX-2 in lung carcinoma. J Exp Med 15
2005;202:931-9. 16
30. Moriwaki K, Bertin J, Gough PJ, Chan FK. A RIPK3-caspase 8 complex mediates atypical 17
pro-IL-1beta processing. J Immunol 2015;194:1938-44. 18
31. Wang JH, Manning BJ, Wu QD, Blankson S, Bouchier-Hayes D, Redmond HP. 19
Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and 20
invasion through a beta 1 integrin-dependent mechanism. J Immunol 2003;170:795-804. 21
32. Banerjee D, Liou HC, Sen R. c-Rel-dependent priming of naive T cells by inflammatory 22
cytokines. Immunity 2005;23:445-58. 23
33. Vlantis K, Wullaert A, Polykratis A, Kondylis V, Dannappel M, Schwarzer R, et al. NEMO 24
Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by 25
NF-kappaB-Dependent and -Independent Functions. Immunity 2016;44:553-67. 26
34. Najjar M, Saleh D, Zelic M, Nogusa S, Shah S, Tai A, et al. RIPK1 and RIPK3 Kinases 27
Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4. Immunity 28
2016;45:46-59. 29
35. Schweitzer K, Bozko PM, Dubiel W, Naumann M. CSN controls NF-kappaB by 30
deubiquitinylation of IkappaBalpha. EMBO J 2007;26:1532-41. 31
36. Weinlich R, Bortoluci KR, Chehab CF, Serezani CH, Ulbrich AG, Peters-Golden M, et al. 32
TLR4/MYD88-dependent, LPS-induced synthesis of PGE2 by macrophages or dendritic cells 33
prevents anti-CD3-mediated CD95L upregulation in T cells. Cell Death Differ 34
2008;15:1901-9. 35
37. Liao X, Lochhead P, Nishihara R, Morikawa T, Kuchiba A, Yamauchi M, et al. Aspirin use, 36
tumor PIK3CA mutation, and colorectal-cancer survival. N Engl J Med 2012;367:1596-606. 37
38. Algra AM, Rothwell PM. Effects of regular aspirin on long-term cancer incidence and 38
metastasis: a systematic comparison of evidence from observational studies versus 39
randomised trials. Lancet Oncol 2012;13:518-27. 40
39. Kumar V, Donthireddy L, Marvel D, Condamine T, Wang F, Lavilla-Alonso S, et al. 41
Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade 42
by Inducing PMN-MDSC Infiltration of Tumors. Cancer Cell 2017;32:654-68 e5. 43
40. Wang D, Wang H, Brown J, Daikoku T, Ning W, Shi Q, et al. CXCL1 induced by 44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
16
prostaglandin E2 promotes angiogenesis in colorectal cancer. J Exp Med 2006;203:941-51. 1
41. Jamieson T, Clarke M, Steele CW, Samuel MS, Neumann J, Jung A, et al. Inhibition of 2
CXCR2 profoundly suppresses inflammation-driven and spontaneous tumorigenesis. J Clin 3
Invest 2012;122:3127-44. 4
42. Wu W, Sun C, Xu D, Zhang X, Shen W, Lv Y, et al. Expression of CXCR2 and its clinical 5
significance in human colorectal cancer. Int J Clin Exp Med 2015;8:5883-9. 6
43. Kaliberova LN, Kusmartsev SA, Krendelchtchikova V, Stockard CR, Grizzle WE, Buchsbaum 7
DJ, et al. Experimental cancer therapy using restoration of NAD+ -linked 8
15-hydroxyprostaglandin dehydrogenase expression. Mol Cancer Ther 2009;8:3130-9. 9
44. Half E, Sun Y, Sinicrope FA. Anti-EGFR and ErbB-2 antibodies attenuate cyclooxygenase-2 10
expression and cooperatively inhibit survival of human colon cancer cells. Cancer Lett 11
2007;251:237-46. 12
13
14
15
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
17
FIGURE LEGENDS 1
Figure 1. RIPK3 is down-regulated in MDSC of CRC tissues. A, Schematic of mice treated 2
with AOM and DSS (Model 1, 2% DSS drinking for 5 days each cycle). The entity image of the 3
colorectal after mice sacrifice is shown on the right panel. B, The percentage of leukocytes 4
(CD45+) in the tumor and colorectal tissue. C, The percentage of MDSC (CD11b
+Gr-1
+) in CD45
+ 5
cells in the tumor and colorectal tissue. D,E, RIPK3 expression in MDSC in the tumor and 6
colorectal tissue by FCM (D) and confocal microscopy (E) (RIPK3 red,Gr-1 green). F, RIPK3 7
expression in MDSC in human colorectal cancer and adjacent tissue. G, After mouse BM cells 8
were treated with PBS or tumor supernatants (from CT26 cells) for 48 hrs, the percentage of 9
CD11b+Gr-1
+ cells in BMCs and RIPK3
+ cells in CD11b
+Gr-1
+ cells were determined. Data of 10
B-G were expressed as mean±SEM. *P<0.05,
**P<0.01, and
****P<0.0001, by Student’s t test. 11
12
Figure 2. RIPK3 deficiency promotes MDSC infiltration and CRC tumorigenesis. A-K, WT 13
and RIPK3-KO (KO) mice model were established with the protocol as Model 1 in Fig. 1A. The 14
body weight were monitored (A), the tumor number (B), colorectal length (C), spleen weight (D), 15
survival (E), MDSC infiltration in tumor by immunofluorescence staining (F), MDSC percentage 16
in tumor (G), colorectal (H) and spleen (I); the percentages of immune cells (J), CD11b+Ly6G
+ 17
and CD11b+Ly6C
+ cells (K) in the tumor tissue upon sacrifice were assessed. Data were expressed 18
as mean±SEM. *P<0.05,
**P<0.01 and ***P<0.001, WT vs. KO, by Student’s t test. 19
20
Figure 3. RIPK3 absence in MDSC enhances the immunosuppressive activity in vitro. A-C, 21
After bone marrow cells from WT and RIPK3 knockout (KO) mice were treated with GM-CSF 22
(20 ng/ml) for 48 hrs, the proportion (A), proliferation (B) and death (C) of MDSC were examined. 23
After treatment with GM-CSF (20 ng/ml) for 48 hrs, the differentiation of WT and KO MDSC 24
into MΦs and DCs (D), as well as the expression of Arg-1, NOS2 and ROS (E,F) were examined. 25
G, CD8+ T cells were co-cultured with WT/RIPK3-KO MDSC (10:1) for 3 days, the proliferation 26
of CD8+ T cells were determined by CFSE. H, After CD8
+ T cells were co-cultured with 27
WT/RIPK3-KO MDSC (10:1) for 48 hrs, the expression of GzmB and IFN-γ were assessed with 28
FCM. I, After MDSC were treated with vehicle (PBS) or NHNL (30 μM), CD8+ T cells were 29
co-cultured with MDSC (5:1) for 48 hrs, and the expression of GzmB and IFN-γ were assessed. J, 30
After CT26 colorectal cancer cells were cultured with conditioned medium (supernatant of 31
WT/RIPK3-KO MDSC : culture medium=1:1) for 48 hrs, the proliferation was assessed. Data 32
were expressed as mean±SEM. *P<0.05,
**P<0.01, and
***P<0.001, by Student’s t test. 33
34
Figure 4. RIPK3 in myeloid derived cells is essential for inhibiting colorectal tumorigenesis. 35
A-I, The chimeric mice and CRC model were established as indicated in Methods. The body 36
weight (A), mortality (B), tumor formation ratio (C), colorectal length (D), spleen weight (E), 37
MDSC percentage in colorectal (F), and MDSC percentage in spleen (G) , g-MDSC percentage in 38
colorectal (H), and g-MDSC percentage in spleen (I) upon sacrifice were assessed. J-O, CRC 39
model of MDSC depletion and CXCL1 receptor CXCR2 antagonist treatment were established as 40
indicated in Methods. The body weight (J), mortality (K), tumor number (L), colorectal length 41
(M), MDSC percentage in colorectal (N) and in spleen (O). Data were expressed as mean±SEM. 42 *P<0.05 and
***P<0.001, by Student’s t test. 43
44
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
18
Figure 5. NF-κB/COX-2/PGE2 signaling is enhanced in RIPK3 deficient MDSC. A, The 1
expression of Gr-1 and COX-2 in tumor and colorectal tissue. B, COX-2 expression in 2
tumor-bearing WT/RIPK3-KO MDSC was evaluated by FCM. C, MRM chromatograms, MS/MS 3
spectrum and production of PGE2 from BM-derived WT/RIPK3-KO MDSC identified with 4
UPLC-MS/MS. D, The expression of COX-2 and p65 in BM-derived WT/RIPK3-KO MDSC. 5
Data were expressed as mean±SEM. *P<0.05,
**P<0.01 and
***P<0.001, by 1-way ANOVA with 6
Sidak’s multiple comparisons test (A) or Student’s t test (B,C). 7
8
Figure 6. Blockade of COX-2 or EP2 attenuates tumorigenesis. A-E, WT and RIPK3-KO mice 9
CRC model 2 (Supplementary Fig. S3F) treated with or without 0.02% ASA containing water 10
drinking as indicated in Methods, the body weight, tumor number, colorectal length, and spleen 11
weight upon sacrifice were determined (A-D). The accumulation of MDSC and COX-2 expression 12
in MDSC of tumor, colorectal and spleen were analyzed (E). F-L, WT and RIPK3-KO Mice CRC 13
model 2 treated with or without AH-6809 (5mg/kg) as indicated in Methods, the body weight (F), 14
tumor number, colorectal length, spleen weight (G-I), and the percentage of MDSC in tumor, 15
colorectal and spleen upon sacrifice were determined (J-L). M,N, After BM-derived MDSC from 16
WT or RIPK3-KO mice were treated with vehicle (PBS), PGE2 (10 μM), ASA (10 μM), or 17
AH6809 (10 μM) for 48 hrs, Arg-1 expression (M) and percentage of differentiation toward M2 18
macrophages (N) were assessed with FCM. O,P, After BM-derived MDSC from WT or 19
RIPK3-KO mice were treated with/without ASA (10 μM), CAPE (2 μM) or AH-6809 (10 μM) for 20
48 hrs, CD8+ T cells were co-cultured with MDSC (16:1) for 24 hrs. The expression of IFN-γ (O) 21
and GzmB (P) in CD8+ T cells were assessed with FCM. Data were expressed as mean±SEM. 22
*P<0.05,
**P<0.01,
***P<0.001 and
****P<0.0001, by Student’s t test (A-L) or 1-way ANOVA with 23
Sidak’s multiple comparisons test (M-P). 24
25
Figure 7. PGE2 negatively regulates RIPK3 and upregulates NF-κB and COX-2 in MDSC 26
and CRC cells. A, BM-derived MDSC were treated with vehicle (PBS) or PGE2 (10 μM) for 48 27
hrs, the expression of RIPK3, p65 and COX-2 were assessed with western blot. B,C, BM-derived 28
MDSC were treated with vehicle (PBS), PGE2 (10 μM), H89 (20 μM) or AH6809 (10 μM) for 48 29
hrs, the expression of PKA, CREB, p-CREB and RIPK3 were determined with western blot (B), 30
the RIPK3 mRNA level was determined with qPCR (C). D, BM-derived MDSC were treated with 31
vehicle (PBS) or ASA (10 μM) for 48 hrs, the protein expression of RIPK3 was assessed. E,F, 32
RIPK3 expression of MDSC from bone marrow cells of WT mice were treated with vehicle (PBS), 33
Bevacizumab (2.5mg/ml), Cetuximab (0.5mg/ml), Nimotuzumab (0.5mg/ml) (E), CPT-11 (300nM), 34
OXA (30nM), 5-FU (250nM) or GEM (100nM) (F). G, Confocal microscopy determination of 35
RIPK3 expression in MDSC in human polypus and cancer tissues at different stages. Data were 36
expressed as mean±SEM. *P<0.05, by 1-way ANOVA with Sidak’s multiple comparisons test. 37
(H,I) Pearson’s correlation coefficient was used to determine the correlation between RIPK3 and 38
indicated genes including CD33, S100A8 (H) and PTGS2 (COX-2) (I) in 148 patients with CRC 39
was examined (GSE21510). J, Patient survival data were obtained from GEO database 40
(GSE17536), overall survival probability were then calculated using the Kaplan-Meier method, 41
and the differences in survival curves were analyzed using the log-rank test. 42
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Published OnlineFirst July 16, 2018.Cancer Res Guifang Yan, Huakan Zhao, Qi Zhang, et al. cell-potentiated colorectal carcinogenesisA RIPK3-PGE2 circuit mediates myeloid-derived suppressor
Updated version
10.1158/0008-5472.CAN-17-3962doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerres.aacrjournals.org/content/suppl/2018/07/13/0008-5472.CAN-17-3962.DC1
Access the most recent supplemental material at:
Manuscript
Authorbeen edited. Author manuscripts have been peer reviewed and accepted for publication but have not yet
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/early/2018/07/14/0008-5472.CAN-17-3962To request permission to re-use all or part of this article, use this link
Research. on September 18, 2020. © 2018 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2018; DOI: 10.1158/0008-5472.CAN-17-3962
Recommended