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The involvement of M2 macrophage polarization inhibition in fenretinide-mediated chemopreventive effects on colon cancer
Accepted Manuscript The Involvement of M2 Macrophage Polarization Inhibition in Fenretinide-mediated Chemopreventive Effects on Colon Cancer Rong Dong, Yanling Gong, Wen Meng, Meng Yuan, Hong Zhu, Meidan Ying, Qiaojun He, Ji Cao, Bo Yang PII:
S0304-3835(16)30717-0
DOI:
10.1016/j.canlet.2016.11.029
Reference:
CAN 13133
To appear in:
Cancer Letters
Received Date: 5 October 2016 Revised Date:
23 November 2016
Accepted Date: 23 November 2016
Please cite this article as: R. Dong, Y. Gong, W. Meng, M. Yuan, H. Zhu, M. Ying, Q. He, J. Cao, B. Yang, The Involvement of M2 Macrophage Polarization Inhibition in Fenretinide-mediated Chemopreventive Effects on Colon Cancer, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.11.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The
Involvement
of
M2
Macrophage
Polarization
Inhibition
in
Fenretinide-mediated Chemopreventive Effects on Colon Cancer
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Rong Dong1, Yanling Gong1, Wen Meng2, Meng Yuan1, Hong Zhu1, Meidan Ying1, Qiaojun He1, Ji Cao1,*, and Bo Yang1,*
1, Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory
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of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang
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University, Hangzhou, China
2, Hangzhou First People's Hospital, Hangzhou, China
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Running title: 4-HPR inhibits macrophages M2 polarization
Key words: 4-HPR, tumor-associated macrophages, cancer prevention,
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STAT6, colon cancer
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Financial support: This work was supported by grants from the National Natural Science Foundation of China (No.81402951 to Ji Cao; No.81473226 to Bo Yang), Zhejiang Provincial Natural Science Foundation (No.LY15H160009 to Wen Meng), Science Technology Department of Zhejiang Province (No.2014C33116 to Wen Meng), and the Fundamental Research Funds for the Central Universities (Ji Cao, Qiaojun He and No.2014XZZZ004 to Bo Yang)
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ACCEPTED MANUSCRIPT *, Corresponding authors: Bo Yang, Room 113, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Email: [email protected]. Ji Cao, Room 115, College of Pharmaceutical Sciences, Zhejiang University,
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Hangzhou, China. Email: [email protected]
Abstract: 161 words
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Figures/tables: 6 figures
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Text: 2916 words
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Reference: 50
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ACCEPTED MANUSCRIPT Abstract Clinical studies have shown that fenretinide (4-HPR) is an attractive chemopreventive agent for cancer treatment. However, to date, few studies
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have demonstrated the mechanism of the preventive effect of 4-HPR. In our current study, we revealed that 4-HPR could significantly suppress IL-4/IL-13 induced M2-like polarization of macrophages, which was demonstrated by the
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reduced expression of M2 surface markers, the down-regulation of M2 marker
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genes, and the inhibition of M2-like macrophages promoted angiogenesis. Mechanistically, our study suggested that the inhibition of the phosphorylation of STAT6, rather than the generation of oxidative stress, is involved in the 4-HPR-driven inhibition of M2 polarization. More intriguingly, by utilizing
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adenomatous polyposis coli (APCmin/+) transgenic mice, we demonstrated that the tumorigenesis was dramatically decreased by 4-HPR treatment accompanied with fewer M2-like macrophages in the tumor tissues, thereby
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profoundly blocking tumor angiogenesis. These findings, for the first time,
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reveal the involvement of M2 polarization inhibition in 4-HPR-mediated chemoprevention, which provides a new point of insight and indicates the potential mechanism underlying the chemopreventive effect of 4-HPR.
Key words: 4-HPR, tumor-associated macrophages, cancer prevention, STAT6, colon cancer
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ACCEPTED MANUSCRIPT 1.Introduction Colorectal cancer (CRC), one of the most high-risk human malignant tumors, is the third most common cancer and the fourth leading cause of
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cancer deaths worldwide, accounting for approximately 1.2 million new cases and 0.6 million deaths per year [1]. Despite advances in the combined use of surgery, radiation, chemotherapy and other therapies, colorectal cancer
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patients still encounter several problems, such as recurrences, low
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postoperative 5-year survival rates and high metastasis rates. The 5-year relative survival for patients diagnosed with metastatic CRC is less than 15% [2]. Given that most cases of colorectal cancer are sporadic and develop slowly over a minimum of 5-10 years through the adenoma-carcinoma
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sequence, early detection and treatment are crucial for the improvement in morbidity and mortality of patients afflicted with this disease[3]. Tumors are composed of an array of cell types, including not only cancer
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cells but also the non-cancer cells. The most prominent component of these
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non-cancer cells are macrophages, which are called tumor-associated macrophages (TAMs)[4, 5]. Usually, monocytes or macrophages undergo profound functional reprogramming in response to various signals, cytokines, and the extreme outcomes of which reflect the plasticity of macrophages. M1/M2 polarized macrophages are extremes of a continuum in a universe of functional states. Classically activated macrophages (M1), alternatively activated macrophages (M2), or both have been observed in tumors[6]. It is
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ACCEPTED MANUSCRIPT generally considered that these two types of macrophages are functionally antagonistic. M2 macrophages are mainly involved in moderating inflammatory responses, promoting angiogenesis and contributing to tissue remodeling, all
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of which are, promote tumor progression[7, 8]. Increasing evidence suggests that some of the key molecules that are altered play significantly important roles in CRC progression and are associated with inflammation[9]. Moreover
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several investigations confirmed significantly higher levels of cathepsin B in
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CRC, which might be associated with the progression of colorectal adenoma to carcinoma [10, 11]. Of interest, evidence has shown that macrophages are the primary source of cathepsins[12]. Thus studying the relationship between macrophage infiltration and colorectal patients’ tumor progression has
results[13].
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attracted intensive interest, but this association has rendered conflicting
Fenretinide (4-HPR), a synthetic derivative of all-trans retinoic acid, can
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cause cancer cell death with the added benefit of having only minor side
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effects [14]. We and other groups have demonstrated that 4-HPR can induce apoptosis in multiple human cancer cell lines, both in vitro and in vivo[15-17]. In clinical trials, 4-HPR has been tested as both a chemopreventive agent in breast, bladder and oral mucosal cancers and a chemotherapeutic agent in pediatric and adult cancers[18-20]. Thus far, a possible benefit was only achieved in premenopausal women regarding the incidence of second breast malignancies of women with breast cancer, a result that persisted in a 15-year
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ACCEPTED MANUSCRIPT follow-up[18]. Given the chemopreventive effect of 4-HPR observed in the clinic, the mechanism of action of 4-HPR prevention of cancer incidence has drawn much attention[21, 22]. However, little progress has been made in this as
the
molecular
mechanisms
chemoprevention remain poorly understood.
underlying
4-HPR-mediated
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area
In the current study, we observed that 4-HPR efficiently skewed
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macrophages away from M2 polarization induced by IL-4/IL-13 in vitro under a
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sub-cytotoxic concentration. Further, we demonstrated that the canonical pathway of STAT6 participated in the inhibition of the M2 polarization by 4-HPR. Additionally, by applying the APCmin/+ transgenic mouse model, we also showed that 4-HPR inhibited colorectal tumorigenesis in vivo through the
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prevention of tumor angiogenesis. Importantly, the in vivo effect of 4-HPR was linked to a decrease in the number of M2-like macrophages in the tumor region. Altogether, for the first time, our study showed that 4-HPR can inhibit TAM M2
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polarization, which might explain the chemopreventive effect of 4-HPR in the
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clinic and open a new avenue to prevent CRC by applying modulators of TAM M2 polarization.
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ACCEPTED MANUSCRIPT 2.Materials and methods 2.1 Ethics statement This investigation has been conducted in accordance with the ethical
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standards and according to the Declaration of Helsinki and national and international guidelines and has been approved by the authors’ institutional review board.
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2.2 Reagents
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4-HPR was kindly provided by Dr.B.J.Maurer (Children’s Hospital, Los Angeles, CA). Recombinant murine IL-4 and IL-13 were purchased from PeproTech, Inc., (210-14 and 200-13 Rocky Hill, NJ, USA). Mouse recombinant M-CSF (300-25-50) and antibody against STAT6 (9362s) were
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purchased from Cell Signalling Technology (Beverly, MA, USA). Antibody against β-actin (sc1615) was purchased from Santa Cruz Biotechnology (CA, USA). Antibodies for p-STAT6 (ab54461) and CD31 (ab28364) were
PE-conjugated
anti-mouse
CD206
(141706),
PE-conjugated
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including
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purchased from Abcam (Cambridge, UK). Antibodies for flow cytometry
anti-mouse CD86 (105008) and FITC-conjugated anti-mouse F4/80 (123108) were purchased from Biolegend (San Diego, CA, USA). The jetPrime transfection agent was obtained from Polyplus (114-15, 850 bd Sébastien Brant 67400 Illkirch FRANCE). 2.3 Cell culture and differentiation RAW264.7, HCT 116, SW620, SW480, and Colo 205 cells were obtained
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ACCEPTED MANUSCRIPT from the Cell Bank of the China Science Academy (Shanghai, China). All the cell lines have been tested and authenticated utilizing short tandem repeat (STR) profiling every 6 months. RAW264.7, HCT 116, SW480, and Colo 205
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were cultured in DMEM, and SW620 was maintained in RPMI-1640 medium, both containing 10% FBS and 100U per ml of penicillin-streptomycin in a 5% CO2 humidified incubator at 37 . RAW264.7 was seeded in 6-well plates at a
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density of 2×105/well and differentiated with 10 ng/ml IL-4/IL-13 for 48 h. To
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examine the effects of 4-HPR, it was added in the medium at the same time with the cytokines.
2.4 Bone marrow derived macrophages isolation and differentiation Bone marrow derived macrophages (BMDMs) were produced as
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previously described with small modification[23]. Briefly, six-week-old C57BL/6 mice were sacrificed and soaked in 75% ethanol. Bone marrow cells were cultured in DMEM containing 10% FBS and 50 ng/ml M-CSF for three days to
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obtain BMDMs.
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2.5 Cell survival assay
For the analysis of cell proliferation, cells were stained by sulforhodamine
B as described previously and evaluated by the multiscan spectrum[24]. The inhibition rate of cell proliferation for each well was calculated. 2.6 Flow cytometry RAW264.7 and BMDMs were collected with a scraper and blocked with 3% BSA for 45 mins, and then were incubated with PE-conjugated anti-mouse
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ACCEPTED MANUSCRIPT CD86 (1:1000), PE-conjugated anti-mouse CD206 (1:1000) antibody or FITC-conjugated anti-mouse F4/80 (1:200), according to the manufacturers’ instructions. For each sample at least 1*104 cells should be analyzed using the
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BD FACS-Calibur cytometer (Becton Dickin-son, San Jose, CA). 2.7 Detection of intracellular ROS
The production of intracellular ROS was measured in the RAW264.7 cell ,
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line using the oxidation-sensitive fluorescent dye DCFH-DA. An increase in
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green fluorescence intensity was used to quantify the generation of intracellular ROS. After DCFH-DA was added at a final concentration of 15.0 µM to the culture medium, the cells were incubated at 37
for indicated time,
and then they were harvested, washed with PBS, and measured immediately
bandpass filter.
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by FACS-Calibur cytometer using an argon laser at 488 nm and a 525-nm
2.8 PI staining for FACS analysis
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The sub-G1 analysis after PI staining was employed to assess apoptosis.
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Cells were treated, harvested and fixed with 75% ethanol at -20°C, then incubated with RNaseA and subsequently with PI in the dark for 30 min. For each sample at least 5 × 104 cells were analyzed using an FACS-Calibur cytometer (Becton Dickinson, San Jose, CA). 2.9 Cell transfection The siRNA sequence duplexes were produced by Genepharma, Co. (Shanghai, China). The siRNAs sequences used were as follows:
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ACCEPTED MANUSCRIPT si-STAT61#: sense: 5’: GCCGAGGCACCCUGUAUAUCCTT , antisense: TTGACGGCUCCGUGGGACAUAUA – 5’; si-STAT62#: sense: 5’: CGAAUGUGAUACAACUGUAUCTT,
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antisense: TTGAGCUUACACUAUGUUGACAU – 5’; Scramble siRNA: sense: 5’: AUCCGCGCGAUAGUACGUATT, antisense: TTUAGGCGCGCUAUCAUGCAU-5’;
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The transfection was performed using siRNA and jetPrime according to the
2.10 Quantitative PCR assay
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manufacturer’s recommendations.
Total RNA from RAW264.7 and BMDMs were extracted from cultured cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the
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manufacturer’s instructions. First-strand cDNA was synthesized with Transcript one-step gDNA Removal and cDNA Synthesis supermix (Transgene Biotech Co., Ltd) and analyzed by real-time quantitative PCR with SYBR Premix Ex (RR420A, TaKaRa, Dalian, China). The reaction mixtures containing
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TaqTM
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SYBR Green were composed following the manufacturer’s protocol. Relative expression levels of the target genes were normalized to control gene 18s rRNA.
The sequences of the primers used for quantitative real time-PCR were as
follows: Fizz: forward-CCCTGCTGGGATGACTGCTA, reverse-TGCAAGTATCTCCACTCTGGATCT;
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ACCEPTED MANUSCRIPT PPAR-γ: forward- TTCGATCCGTAGAAGCCGTG, reverse- TTGGCCCTCTGAGATGAGGA; 18s rRNA: forward- CGGCTACCACATCCAAGGAA,
2.11 Conditioned medium preparation
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reverse- GCTGGAATTACCGCGGCT.
Macrophage polarization was obtained by culturing cells in DMEM
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medium supplemented with 10% FBS and 10 ng/ml IL4/IL13 or 5µM 4-HPR for
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three days. Then different polarized RAW264.7 cells were incubated in serum free medium for 24h, after which culture supernatants were collected as conditioned medium (CM). CM was centrifuged at 3000 rpm to separate out the debris and stored at -80
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2.12 Tube formation assay
HUVEC cells (3x104 cells per well) were seeded in 96-well plates that were filled with 50µl Matrigel and solidified in 37
. The cells were cultured in
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CM supplemented with 2.5% FBS for 6 hours. To observe the formation of
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tube-like structures, five optical fields (10x magnification) per well were randomly chosen and analyzed by a LEICA DMI 4000B microscope with Leica Application Suite software. 2.13 Western blot analysis After treatment with compounds for the indicated times, the macrophages were harvested. Cells were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM β-Sodium
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ACCEPTED MANUSCRIPT Glycerophosphate, 0.3% NP-40, 0.3% Triton X-100, 0.25% Leupeptin, 0.1% PMSF, 0.1% NaVO3). Cell lysates were centrifuged at 13,200 rpm for 30 min at 4
. Proteins were separated in 8% SDS-PAGE and blotted onto PVDF
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membranes, then incubated with primary antibodies followed by HRP-labelled secondary antibodies. The protein level was then detected using an ECL-plus kit and visualized on autoradiography film.
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2.14 Animals
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APCmin/+ mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The following primers were used for genotyping:
APC (wild type mice) Forward: 5’-GCCATCCCTTCACGTTAG-3’
APC
(for
both
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APC (APCmin/+ mice) Forward: 5’-TTCTGAGAAAGACAGAAGTTA-3’ wild
type
and
APCmin/+
mice)
Reverse:
5’-TTCCACTTTGGCATAAGGC-3’.
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Both genders were used in the experiments unless specifically indicated.
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The mice were housed under standard light/dark cycling and had access to food and water ad libitum. All the protocols were approved by the local ethics committee. To evaluate in vivo tumorigenesis, 6-week-old APCmin/+ mice were fed with a HFD (45% fat, Xietong organism, Nanjing, China). 2.15 TUNEL assay The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was used to evaluate the apoptotic response of tumor tissues
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ACCEPTED MANUSCRIPT in formalin-fixed tumor samples in paraffin blocks using the one-step TUNEL Apoptosis Assay kit (C1088, Beyotime, Nanjing, China) according to the manufacturer’s instructions.
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2.16 Histology and immunohistochemistry Tissues were fixed in 4% formaldehyde and embedded in paraffin. Then 3-5µm
sections
from
all
the
tissues
were
cut
and
stained
with
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hematoxylin/eosin (H&E) and reticulin. All the sections were reviewed by a
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pathologist. Immunohistochemistry was performed using an automated immunostainer with a DAB detection kit according to the company’s protocols for open procedures with slight modifications, using mouse monoclonal anti-CD206 (dilution 1:200, ab64693, Abcam, Cambridge, UK), anti-CD31
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(dilution 1:200, ab28364, Abcam, Cambridge, UK) and anti-Ki67 (dilution 1:50, sc-15402, Santa Cruz Biotechnology CA, USA). For antigen retrieval, sections were boiled in EDTA buffer (pH 9) for 30min prior to staining.
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2.17 Statistical analysis
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Values are presented as means ± SD. Two-tailed and unpaired Student’s t-test was used for statistical analysis, and differences were considered significant for p values less than 0.05.
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ACCEPTED MANUSCRIPT 3. Results 3.1 4-HPR efficiently inhibits M2 polarization of macrophages induced by IL-4/IL-13
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Because M2 TAMs mainly contribute to the poor progression of tumors, and 4-HPR has been shown to be a chemopreventive agent in clinical trials[25], it is interesting to assess whether 4-HPR could affect the M2 polarization of
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macrophages. IL-4/IL-13 induces a distinct functional and phenotypic
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response of macrophages often described as M2 macrophage polarization (compared to M1 polarization describing a macrophage phenotype in response to the Th1 cytokine IFN-γ or toll-like receptor (TLR) activation)[26, 27]. Therefore we first analyzed the impact of 4-HPR on IL-4/IL-13 induced M2-like
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polarization of macrophages in vitro. As expected, significant up-regulation of CD206 was observed when RAW264.7 cells were treated with either 10 ng/ml IL-4 or IL-13 for 48h (Figure 1A and 1B). Meanwhile, the expression of CD206
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induced by IL-4/IL-13 was greatly reduced by additionally treatment with 5µM
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4-HPR (from 28.61±4.08% to 11.48±2.41% and 33.29±2.55% to 14.21±2.69%, respectively) (Figure 1A and 1B). Since polarization of macrophages is accompanied by profound gene expression changes, we next measured the expression of specific M2 TAM marker genes, Fizz1 and PPAR-γ[28]. A significant decrease of these two genes was observed after co-administration of 5µM 4-HPR and IL-4/IL-13 compared with the IL-4/IL-13 only group (Figure 1C). The inhibition of M2 polarization is not caused by the 4-HPR-mediated
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ACCEPTED MANUSCRIPT cytotoxicity (Figure S1A and S1B). To further validate the effect of 4-HPR on M2 polarization, we generated primary bone marrow derived macrophages (BMDMs) from mice and performed the same experiments. Again, IL-4/IL-13
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induced CD206 expression in BMDMs was totally blocked by 4-HPR (Figure 1D and 1E), and the decrease of M2 marker genes also confirmed the inhibition of M2 TAM polarization caused by 4-HPR (Figure 1F). Of note,
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4-HPR treatment did not block the M1 polarization in RAW264.7 cells caused
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by IFN-γ and lipopolysaccharides (LPS) (Figure S1C). Therefore, these results clearly suggest that 4-HPR can effectively and specifically inhibit the M2 polarization of macrophages in vitro.
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3.2 ROS is not involved in the 4-HPR-mediated inhibition of the M2 polarization of macrophages
We and other groups have proven that the generation of reactive oxygen
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species (ROS) mediates 4-HPR tumor cell cytotoxicity[15, 17, 29]. Thus, it is
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necessary to study whether ROS was also involved in 4-HPR-mediated inhibition of TAM M2 polarization. To address this issue, we first analyzed the ROS level of RAW264.7 cells treated with 4-HPR. As shown in Figure 2A, a significant generation of ROS was observed when the RAW264.7 cells were treated with 5µM 4-HPR. To investigate the relationship between the inhibition of M2 polarization and ROS, we applied the ROS scavengers N-acetylcysteine (NAC) and glutathione (GSH) to remove intracellular ROS generated in
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ACCEPTED MANUSCRIPT response to 4-HPR treatment. Intriguingly, the NAC and GSH promoted the inhibition ratio of M2 polarization of macrophages treated with 4-HPR, which indicated that ROS might promote rather than inhibit the M2 polarization
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(Figure 2B). In order to confirm the general role of ROS in M2 polarization, we next used H2O2 to generate ROS and test the impact of H2O2 on the M2 polarization. As shown in Figure 2C and 2D, H2O2 alone or together with
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IL-4/IL-13 could induce or promote CD206 expression. Moreover, the increase
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of CD206 expression in macrophages with IL-4/IL-13 and H2O2 could be reversed by NAC and GSH (Figure 2E). Taken together, these results suggest that 4-HPR mediated the inhibition of TAMs M2 polarization in an
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ROS-independent manner.
3.3 The STAT6 signaling pathway participates in 4-HPR-mediated inhibition of M2 polarization
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The classical pathway for IL-4-induced TAM polarization involves the
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activation of the STAT6 signaling pathway[30]. Once STAT6 is activated, it can bind directly to the promoter of certain downstream genes such as Fizz1 and transcriptionally activates these genes[31]. As 4-HPR inhibited M2 polarization specific gene Fizz1 gene expression, we next assessed whether STAT6 participated in 4-HPR-mediated inhibition of M2 macrophage polarization. In general, STAT6 is activated rapidly upon cytokine receptor binding. IL-4 can sustain the phosphorylation level of STAT6 during the polarization process.
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ACCEPTED MANUSCRIPT Thus we first investigated the phosphorylation of STAT6 after treatment with IL-4/IL-13 with or without 4-HPR. As shown in Figure 3A and 3C, in RAW264.7 and BMDM cells, the phosphorylation status of STAT6 was increased after
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treating cells with IL-4/IL-13 for 2h. Meanwhile the increase of phosphorylated STAT6 was dramatically inhibited by co-treatment with 4-HPR, which suggests that STAT6 might participate in 4-HPR-mediated inhibition of TAMs M2
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polarization (Figure 3B and 3D). To further confirm this result, we applied the
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siRNA to specifically knockdown the intracellular protein expression of STAT6, and then measured the change of the inhibition of M2 polarization caused by 4-HPR. As shown in Figure 3E, two siRNAs against STAT6 remarkably decreased the endogenous STAT6 expression in cells. Moreover, the STAT6
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silencing by siRNA also significantly reduced the M2-like polarization induced by IL-4/IL-13 as expected. Of note, the blockade of STAT6 expression did not further increase the inhibitory ratio of CD206 caused by 4-HPR (Figure 3F),
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which suggested that inhibition of the phosphorylation of STAT6 significantly
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affected 4-HPR-inhibited M2-like polarization. Therefore, our data support the hypothesis that the STAT6 signaling pathway participates in 4-HPR-mediated inhibition of M2 polarization.
3.4
4-HPR
abrogates
the
angiogenesis
phenotype
of
M2-like
macrophages The next key question is whether 4-HPR-mediated inhibition of M2
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ACCEPTED MANUSCRIPT polarization could result in the inability of tumors [32]. In order to address this issue, we first induced the inhibitory effect of 4-HPR directly on three colorectal cell lines, HCT116, SW620 and Colo 205, to rule out the possibility that
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inhibition of M2 polarization is dispensable for 4-HPR-driven chemoprevention. As shown in Figure S2A, the proliferation of cells treated with 4-HPR was not significantly inhibited compared to those treated with 5-Fluorouracil (5-Fu),
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which is a first-line treatment for colorectal cancer. In addition, no significant
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apoptosis was observed after the treatment with 4-HPR for 72h compared with the treatment with 5-Fu (Figure S2B).
Next, we focused on the two well-known phenotypes of M2 TAMs proliferation promotion and angiogenesis promotion[33, 34]. Macrophages
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were treated with IL-4/IL-13, 4-HPR, or both for 72h, and the culture medium was replaced with fresh medium without serum. Then, 24 h later the supernatant medium was collected (referred to as CM). To evaluate the impact
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of CM on tumor cell survival, the colorectal cell lines HCT116, SW620, and
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SW480 were treated with the CM for 72h and cell proliferation and apoptosis were further analyzed. Surprisingly, no significant difference was found in the IL-4 combined 4-HPR group (vs. the IL-4 group), or the IL-13 combined 4-HPR group
(vs.
the
IL-13
group)
(Figure
4A
and
4B).
For
the
angiogenesis-promoting effect, we evaluated the tube formation of HUVEC cells treated with CM. HUVEC cells were seeded in a 96-well plate pre-coated with Matrigel and formed capillary-like structures in the presence of different
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ACCEPTED MANUSCRIPT supernatants of macrophages. As shown in Figure 4C, the network of tube-like structures in the IL-4/IL-13 group was more extensive than those in the IL-4/IL-13 combined with 4-HPR groups (yellow arrow indicated, Figure 4D).
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Together with the inhibition of M2 polarization we described previously, these data suggest that the subcytotoxic concentration of 4-HPR could abrogate the
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physiological angiogenesis-promoting function of M2 TAMs.
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3.5 4-HPR prevents the colorectal cancer formation in vivo by targeting macrophages
In order to validate our result with an in vivo approach, we then tested whether 4-HPR blocks tumor formation as well as inhibits the occurrence of
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M2 TAMs in APCmin/+ transgenic mice, which are highly susceptible to spontaneous intestinal adenoma formation[35]. Considering that monocytes are constantly produced from bone marrow, we hypothesized that continuous
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4-HPR treatment is necessary to block the differentiation of monocytes into
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TAMs. Six-week-old APCmin/+ mice were maintained on 4-HPR (20mg/kg) for 14 weeks together with a high fat diet. Whole intestines from these mice were collected, and histopathological examination of H&E-stained sections of the intestines was completed in all the mice (Figure 5A). Of note, the number of intestinal tumors was significantly reduced in the 4-HPR treated group as compared to the control group, from 14.33+7.73 to 6.11+7.00 (p<0.01) (Figure 5B and 5C), without affecting the body weight of the mice (Figure 5D).
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ACCEPTED MANUSCRIPT To explore whether TAMs played a critical role in 4-HPR-inhibited tumorigenesis, we then tested if 4-HPR altered TAM polarization in tumor tissues. Immunohistochemistry analysis showed that 4-HPR inhibited TAM M2
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polarization, as less CD206 in 4-HPR treated tumors was detected in the tumor region (Figure 6A). Similar results are shown in Figure 6B, which were determined using log IOD in Image-Pro Plus 6.0, indicating that the expression
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of CD206 was correlated with the tumorigenesis of APCmin/+ mice. Since the
support
the
fact
that
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status of tumor vessels contributes to tumorigenesis and our in vitro data 4-HPR
abolished
the
physiological
angiogenesis-promoting function of M2 TAMs, we tested if 4-HPR affected vessel density in mice. As shown in Figure 6G and 6H, CD31 staining revealed
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that tumor vessel density was dramatically decreased in tumors treated with 4-HPR. Moreover, we also investigated whether 4-HPR effected on the tumor growth and apoptosis. As shown in Figure 6C and 6E, 4-HPR did not induce
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colorectal tumor cell death nor inhibit the cancer cell growth.
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Collectively, these observations suggest that the M2-like phenotype plays a positive role in regulating tumorigenesis in colorectal cancer; nevertheless, 4-HPR could antagonize the tumorigenesis of colorectal cancer induced by M2-like macrophages.
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ACCEPTED MANUSCRIPT 4.Discussion Alhough 4-HPR has received great attention as a chemopreventive agent, based on chemoprevention clinical trials, the underlying mechanism of its
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chemopreventive effect is still poorly understood. Using RAW264.7, we demonstrated the remarkable inhibition of M2 macrophage polarization by the administration of 4-HPR. It should be emphasized that the effective
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concentration of 4-HPR for M2 macrophage polarization inhibition is lower than
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the concentration used for tumor cytotoxicity, which might be a more reasonable mechanism to explain its chemopreventive activity in the clinic. Importantly, although there are several clinical trials of 4-HPR as a cancer chemoprevention agent, none have identified the potential biomarkers in the
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inclusion criteria. Specifically, our finding that M2 macrophages are dramatically decreased in APCmin/+ transgenic mice further suggests that the analysis of an M2 polarization marker in tumor biopsy sections may identify the
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biomarker of the chemopreventive effect of 4-HPR.
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We and other groups have previously reported that 4-HPR inhibited tumor growth and metastasis in animals and functioned as an apoptosis inducer mostly through the production of ROS and mitochondrial disruption[16, 17, 36]. When we treat RAW264.7 cells with H2O2 alone or together with IL-4/IL-13, it could induce or promote CD206 expression, suggesting the positive role of ROS in M2 polarization. These results are consistent with a previous report that ROS elimination by butylated hydroxyanisole and other ROS inhibitors
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ACCEPTED MANUSCRIPT blocks M2 macrophage polarization[37]. However, according to our finding ROS are not involved in 4-HPR-induced inhibition of M2 macrophages. Instead, we found that 4-HPR-induced inhibition of M2 macrophages correlates with
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decreased phosphorylation status of STAT6. Although the underlying mechanism of 4-HPR-triggered inhibition of STAT6 is unknown, based on the fact that 1) ceramide could active several signaling pathways including the
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JAK/STAT axis[38], and 2) recent in vitro data indicates that 4-HPR disrupts
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the biosynthesis of ceramides through the inhibition of dihydroceramide desaturase (Des1)[39], we propose that the inhibition of ceramide synthesis might mediate 4-HPR-triggered inhibition of STAT6.
Two well-known phenotypes of M2 TAMs are proliferation-promotion and
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angiogenesis-promotion[40, 41]. However, in the current study, we did not observe the proliferation promoting effect of M2 macrophages on three colon cancer cell lines, which might explain why we failed to detect a difference in
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colon cancer cell proliferation in the presence of conditioned media derived
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from 4-HPR-treated M2 macrophages. Alternatively, both in vitro and in vivo data suggest that 4-HPR does abrogate the angiogenesis phenotype of M2-like macrophages. Interestingly, previous reports also have suggested that the chemopreventive effects of 4-HPR in early intervention protocols are likely due to its anti-angiogenesis properties. Macrophages represent 10%-20% of all mononuclear cells found in the intestinal lamina propria, making the intestine the largest reservoir of
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ACCEPTED MANUSCRIPT macrophages in humans[42, 43]. The identification of cancer-induced macrophages differentiation characteristics is helpful for understanding the correlation between TAMs and colorectal cancer[44, 45]. The immunological
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role of TAMs in the prognosis of CRC patients has been debated for the past few decades[46, 47]. The inconsistency among these studies is likely due to the cancer characteristics, the preparation of the macrophages studied, and
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the definition of M1/M2 macropahges. Understanding the early events in
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colorectal carcinogenesis is still incomplete. Accumulating evidence has indicated that inflammatory responses are important for cancer development. Epidemiological studies have shown that regular use of non-steroidal anti-inflammatory drugs reduces the risk of colorectal cancer development[48].
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In the tumor microenvironment, tumor-associated macrophages express pro-inflammatory cytokines, including CCL2, IL-4, IL-13, etc. Recent studies have uncovered the role of such an inflammatory network in the promotion of
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gastrointestinal tumor development[13, 49]. Genetically engineered and
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chemically induced mouse tumor models, which mimic sporadic or inflammation-associated tumorigenesis were used in these studies. In our study, we first demonstrated that macrophages might be involved in colorectal cancer chemoprevention. Although several studies revealed that TAMs are a biomarker of colorectal cancer prognosis, none of them ever clearly investigated the relationship between the inhibition of TAMs and tumorigenesis of colorectal cancer[50].
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ACCEPTED MANUSCRIPT In summary, we identify 4-HPR as selectively inhibiting M2 macrophage polarization through the inhibition of phosphorylation of STAT6, and in turn preventing the tumorigenesis of colorectal cancer. Macrophage modulation
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might therefore contribute to the chemopreventive effect of 4-HPR. Incomplete inhibition of macrophage activation during 4-HPR treatment indicates that more efficient strategies are needed to achieve a robust modulation of
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macrophages in the tumor environment and further improve tissue response.
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Our data suggest that specifically addressing the STAT6 signaling pathway is a promising approach to modulate the CRC environment.
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ACCEPTED MANUSCRIPT Figure Legends Figure 1: 4-HPR inhibits M2 polarization of macrophages induced by IL-4/IL-13 (A and B) RAW264.7 cells were exposed to IL-4/IL-13 (10 ng/ml)
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with or without 4-HPR (5 µM) for 3 days and harvested for cell surface marker analysis by flow cytometry. (C) mRNA expression of Fizz1 and PPAR-γ in RAW264.7 was quantified by qRT-PCR and normalized to 18s rRNA
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expression. (D and E) Flow cytometric analysis of CD206 expression
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percentage of BMDMs. (F) mRNA expression of Fizz1 and PPAR-γ in BMDMs was quantified by qRT-PCR and normalized to 18s rRNA expression. Each experiment was repeated three times.
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Figure 2: 4-HPR inhibits M2 polarization of macrophages in a ROSindependent manner. (A) RAW 264.7 cells were treated with 4-HPR for the indicated time and then harvested for DCFH-DA analysis by flow cytometry. (B)
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RAW 264.7 cells were treated with IL-4/IL-13, 4-HPR (5 µM), NAC (2 mM) or
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GSH (4 mM) for 3 days and cells were harvested for analysis of CD206 by flow cytometry. (C and D) RAW 264.7 cells were exposed to IL-4/IL-13 with or without H2O2 (40 µM) for 3 days and then harvested for CD206 analysis by flow cytometry. (E) RAW 264.7 cells were treated with IL-4/IL-13, H2O2 (40 µM), NAC (2 mM) or GSH (4 mM) for 3 days and cells were harvested for CD206 analysis by flow cytometry. Each experiment was repeated at least three times.
ACCEPTED MANUSCRIPT Figure 3: The canonical STAT6 pathway was involved in 4-HPR-induced inhibition of M2-like polarization of macrophages. (A and C) RAW264.7 cells and BMDMs were treated with IL-4/IL-13 with or without 4-HPR (5 µM) for
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2 h. Western blot of STAT6, activated p-STAT6 in cell lysates. Actin served as a loading control. (B and D) Quantification of the p-STAT6/STAT6 at the indicated conditions. (E) RAW264.7 cells were transfected with siRNAs
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specifically targeting STAT6 or control siRNAs, as described in the Materials
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and Methods. Proteins were detected by western blot analysis using specific antibodies. (F) RAW 264.7 cells were harvested and analyzed for CD206 (n=3).
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Figure 4: 4-HPR abrogates the angiogenesis phenotype of M2-like macrophages. (A)Proliferation of colorectal cancer cells (HCT116, SW620, and SW480) in the presence of conditioned media derived from 4-HPR-treated
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or untreated M2 macrophages. Cell growth kinetics over a period of 5 days
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was assessed by SRB assay. (B) The colorectal cancer cells (HCT116, SW620 and SW480) were incubated with conditioned medium for 3 days and the cells were collected for PI analysis by flow cytometry (n=3). (C and D) HUVEC cells were formed capillary-like structures in the presence of the indicated conditioned medium.
Figure 5: 4-HPR prevented colorectal cancer in vivo. (A) Schematic
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groups was measured during the experiment.
Figure 6: 4-HPR prevented colorectal cancer in vivo by targeting (A)
Representative
histopathological
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macrophages.
and
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immunohistochemical image of CRC. Hematoxylin & eosin (H&E) and CD206 stains indicate tumor borders. Macrophages accumulated in colorectal tumor regions, as indicated by CD206. (B) The expression scores of CD206 in the colorectal tissues were analyzed by Image-Pro Plus 6.0. (C and D)
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Representative histopathology and immunohistochemistry of resected CRC. Hematoxylin & eosin (H&E) and TUNEL staining indicated the apoptotic regions. The mean density of the immunofluorescence was quantified by
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Image-J. (E and F) Representative immunohistochemistry of Ki67 in a CRC
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section and the expression scores of Ki67 were analyzed by Image-Pro Plus 6.0. (G and H) Representative immunohistochemistry CD31 stains in tumor borders. Quantification of CD31 positive cells in tissue sections with CRC.
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Highlights 4-HPR could significantly suppress M2-like polarization of macrophages. The inhibition of phosphorylation of STAT6, rather than the generation of oxidative stress, is involved in the 4-HPR-driven inhibition of M2 polarization. The inhibition of M2 polarization in involved in 4-HPR-mediated chemoprevention
S0304-3835(16)30717-0
DOI:
10.1016/j.canlet.2016.11.029
Reference:
CAN 13133
To appear in:
Cancer Letters
Received Date: 5 October 2016 Revised Date:
23 November 2016
Accepted Date: 23 November 2016
Please cite this article as: R. Dong, Y. Gong, W. Meng, M. Yuan, H. Zhu, M. Ying, Q. He, J. Cao, B. Yang, The Involvement of M2 Macrophage Polarization Inhibition in Fenretinide-mediated Chemopreventive Effects on Colon Cancer, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.11.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT The
Involvement
of
M2
Macrophage
Polarization
Inhibition
in
Fenretinide-mediated Chemopreventive Effects on Colon Cancer
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Rong Dong1, Yanling Gong1, Wen Meng2, Meng Yuan1, Hong Zhu1, Meidan Ying1, Qiaojun He1, Ji Cao1,*, and Bo Yang1,*
1, Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory
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of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang
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University, Hangzhou, China
2, Hangzhou First People's Hospital, Hangzhou, China
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Running title: 4-HPR inhibits macrophages M2 polarization
Key words: 4-HPR, tumor-associated macrophages, cancer prevention,
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STAT6, colon cancer
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Financial support: This work was supported by grants from the National Natural Science Foundation of China (No.81402951 to Ji Cao; No.81473226 to Bo Yang), Zhejiang Provincial Natural Science Foundation (No.LY15H160009 to Wen Meng), Science Technology Department of Zhejiang Province (No.2014C33116 to Wen Meng), and the Fundamental Research Funds for the Central Universities (Ji Cao, Qiaojun He and No.2014XZZZ004 to Bo Yang)
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ACCEPTED MANUSCRIPT *, Corresponding authors: Bo Yang, Room 113, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Email: [email protected]. Ji Cao, Room 115, College of Pharmaceutical Sciences, Zhejiang University,
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Hangzhou, China. Email: [email protected]
Abstract: 161 words
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Figures/tables: 6 figures
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Text: 2916 words
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Reference: 50
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ACCEPTED MANUSCRIPT Abstract Clinical studies have shown that fenretinide (4-HPR) is an attractive chemopreventive agent for cancer treatment. However, to date, few studies
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have demonstrated the mechanism of the preventive effect of 4-HPR. In our current study, we revealed that 4-HPR could significantly suppress IL-4/IL-13 induced M2-like polarization of macrophages, which was demonstrated by the
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reduced expression of M2 surface markers, the down-regulation of M2 marker
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genes, and the inhibition of M2-like macrophages promoted angiogenesis. Mechanistically, our study suggested that the inhibition of the phosphorylation of STAT6, rather than the generation of oxidative stress, is involved in the 4-HPR-driven inhibition of M2 polarization. More intriguingly, by utilizing
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adenomatous polyposis coli (APCmin/+) transgenic mice, we demonstrated that the tumorigenesis was dramatically decreased by 4-HPR treatment accompanied with fewer M2-like macrophages in the tumor tissues, thereby
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profoundly blocking tumor angiogenesis. These findings, for the first time,
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reveal the involvement of M2 polarization inhibition in 4-HPR-mediated chemoprevention, which provides a new point of insight and indicates the potential mechanism underlying the chemopreventive effect of 4-HPR.
Key words: 4-HPR, tumor-associated macrophages, cancer prevention, STAT6, colon cancer
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ACCEPTED MANUSCRIPT 1.Introduction Colorectal cancer (CRC), one of the most high-risk human malignant tumors, is the third most common cancer and the fourth leading cause of
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cancer deaths worldwide, accounting for approximately 1.2 million new cases and 0.6 million deaths per year [1]. Despite advances in the combined use of surgery, radiation, chemotherapy and other therapies, colorectal cancer
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patients still encounter several problems, such as recurrences, low
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postoperative 5-year survival rates and high metastasis rates. The 5-year relative survival for patients diagnosed with metastatic CRC is less than 15% [2]. Given that most cases of colorectal cancer are sporadic and develop slowly over a minimum of 5-10 years through the adenoma-carcinoma
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sequence, early detection and treatment are crucial for the improvement in morbidity and mortality of patients afflicted with this disease[3]. Tumors are composed of an array of cell types, including not only cancer
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cells but also the non-cancer cells. The most prominent component of these
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non-cancer cells are macrophages, which are called tumor-associated macrophages (TAMs)[4, 5]. Usually, monocytes or macrophages undergo profound functional reprogramming in response to various signals, cytokines, and the extreme outcomes of which reflect the plasticity of macrophages. M1/M2 polarized macrophages are extremes of a continuum in a universe of functional states. Classically activated macrophages (M1), alternatively activated macrophages (M2), or both have been observed in tumors[6]. It is
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ACCEPTED MANUSCRIPT generally considered that these two types of macrophages are functionally antagonistic. M2 macrophages are mainly involved in moderating inflammatory responses, promoting angiogenesis and contributing to tissue remodeling, all
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of which are, promote tumor progression[7, 8]. Increasing evidence suggests that some of the key molecules that are altered play significantly important roles in CRC progression and are associated with inflammation[9]. Moreover
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several investigations confirmed significantly higher levels of cathepsin B in
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CRC, which might be associated with the progression of colorectal adenoma to carcinoma [10, 11]. Of interest, evidence has shown that macrophages are the primary source of cathepsins[12]. Thus studying the relationship between macrophage infiltration and colorectal patients’ tumor progression has
results[13].
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attracted intensive interest, but this association has rendered conflicting
Fenretinide (4-HPR), a synthetic derivative of all-trans retinoic acid, can
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cause cancer cell death with the added benefit of having only minor side
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effects [14]. We and other groups have demonstrated that 4-HPR can induce apoptosis in multiple human cancer cell lines, both in vitro and in vivo[15-17]. In clinical trials, 4-HPR has been tested as both a chemopreventive agent in breast, bladder and oral mucosal cancers and a chemotherapeutic agent in pediatric and adult cancers[18-20]. Thus far, a possible benefit was only achieved in premenopausal women regarding the incidence of second breast malignancies of women with breast cancer, a result that persisted in a 15-year
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ACCEPTED MANUSCRIPT follow-up[18]. Given the chemopreventive effect of 4-HPR observed in the clinic, the mechanism of action of 4-HPR prevention of cancer incidence has drawn much attention[21, 22]. However, little progress has been made in this as
the
molecular
mechanisms
chemoprevention remain poorly understood.
underlying
4-HPR-mediated
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area
In the current study, we observed that 4-HPR efficiently skewed
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macrophages away from M2 polarization induced by IL-4/IL-13 in vitro under a
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sub-cytotoxic concentration. Further, we demonstrated that the canonical pathway of STAT6 participated in the inhibition of the M2 polarization by 4-HPR. Additionally, by applying the APCmin/+ transgenic mouse model, we also showed that 4-HPR inhibited colorectal tumorigenesis in vivo through the
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prevention of tumor angiogenesis. Importantly, the in vivo effect of 4-HPR was linked to a decrease in the number of M2-like macrophages in the tumor region. Altogether, for the first time, our study showed that 4-HPR can inhibit TAM M2
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polarization, which might explain the chemopreventive effect of 4-HPR in the
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clinic and open a new avenue to prevent CRC by applying modulators of TAM M2 polarization.
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ACCEPTED MANUSCRIPT 2.Materials and methods 2.1 Ethics statement This investigation has been conducted in accordance with the ethical
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standards and according to the Declaration of Helsinki and national and international guidelines and has been approved by the authors’ institutional review board.
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2.2 Reagents
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4-HPR was kindly provided by Dr.B.J.Maurer (Children’s Hospital, Los Angeles, CA). Recombinant murine IL-4 and IL-13 were purchased from PeproTech, Inc., (210-14 and 200-13 Rocky Hill, NJ, USA). Mouse recombinant M-CSF (300-25-50) and antibody against STAT6 (9362s) were
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purchased from Cell Signalling Technology (Beverly, MA, USA). Antibody against β-actin (sc1615) was purchased from Santa Cruz Biotechnology (CA, USA). Antibodies for p-STAT6 (ab54461) and CD31 (ab28364) were
PE-conjugated
anti-mouse
CD206
(141706),
PE-conjugated
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including
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purchased from Abcam (Cambridge, UK). Antibodies for flow cytometry
anti-mouse CD86 (105008) and FITC-conjugated anti-mouse F4/80 (123108) were purchased from Biolegend (San Diego, CA, USA). The jetPrime transfection agent was obtained from Polyplus (114-15, 850 bd Sébastien Brant 67400 Illkirch FRANCE). 2.3 Cell culture and differentiation RAW264.7, HCT 116, SW620, SW480, and Colo 205 cells were obtained
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ACCEPTED MANUSCRIPT from the Cell Bank of the China Science Academy (Shanghai, China). All the cell lines have been tested and authenticated utilizing short tandem repeat (STR) profiling every 6 months. RAW264.7, HCT 116, SW480, and Colo 205
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were cultured in DMEM, and SW620 was maintained in RPMI-1640 medium, both containing 10% FBS and 100U per ml of penicillin-streptomycin in a 5% CO2 humidified incubator at 37 . RAW264.7 was seeded in 6-well plates at a
SC
density of 2×105/well and differentiated with 10 ng/ml IL-4/IL-13 for 48 h. To
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examine the effects of 4-HPR, it was added in the medium at the same time with the cytokines.
2.4 Bone marrow derived macrophages isolation and differentiation Bone marrow derived macrophages (BMDMs) were produced as
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previously described with small modification[23]. Briefly, six-week-old C57BL/6 mice were sacrificed and soaked in 75% ethanol. Bone marrow cells were cultured in DMEM containing 10% FBS and 50 ng/ml M-CSF for three days to
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obtain BMDMs.
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2.5 Cell survival assay
For the analysis of cell proliferation, cells were stained by sulforhodamine
B as described previously and evaluated by the multiscan spectrum[24]. The inhibition rate of cell proliferation for each well was calculated. 2.6 Flow cytometry RAW264.7 and BMDMs were collected with a scraper and blocked with 3% BSA for 45 mins, and then were incubated with PE-conjugated anti-mouse
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ACCEPTED MANUSCRIPT CD86 (1:1000), PE-conjugated anti-mouse CD206 (1:1000) antibody or FITC-conjugated anti-mouse F4/80 (1:200), according to the manufacturers’ instructions. For each sample at least 1*104 cells should be analyzed using the
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BD FACS-Calibur cytometer (Becton Dickin-son, San Jose, CA). 2.7 Detection of intracellular ROS
The production of intracellular ROS was measured in the RAW264.7 cell ,
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line using the oxidation-sensitive fluorescent dye DCFH-DA. An increase in
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green fluorescence intensity was used to quantify the generation of intracellular ROS. After DCFH-DA was added at a final concentration of 15.0 µM to the culture medium, the cells were incubated at 37
for indicated time,
and then they were harvested, washed with PBS, and measured immediately
bandpass filter.
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by FACS-Calibur cytometer using an argon laser at 488 nm and a 525-nm
2.8 PI staining for FACS analysis
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The sub-G1 analysis after PI staining was employed to assess apoptosis.
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Cells were treated, harvested and fixed with 75% ethanol at -20°C, then incubated with RNaseA and subsequently with PI in the dark for 30 min. For each sample at least 5 × 104 cells were analyzed using an FACS-Calibur cytometer (Becton Dickinson, San Jose, CA). 2.9 Cell transfection The siRNA sequence duplexes were produced by Genepharma, Co. (Shanghai, China). The siRNAs sequences used were as follows:
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ACCEPTED MANUSCRIPT si-STAT61#: sense: 5’: GCCGAGGCACCCUGUAUAUCCTT , antisense: TTGACGGCUCCGUGGGACAUAUA – 5’; si-STAT62#: sense: 5’: CGAAUGUGAUACAACUGUAUCTT,
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antisense: TTGAGCUUACACUAUGUUGACAU – 5’; Scramble siRNA: sense: 5’: AUCCGCGCGAUAGUACGUATT, antisense: TTUAGGCGCGCUAUCAUGCAU-5’;
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The transfection was performed using siRNA and jetPrime according to the
2.10 Quantitative PCR assay
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manufacturer’s recommendations.
Total RNA from RAW264.7 and BMDMs were extracted from cultured cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the
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manufacturer’s instructions. First-strand cDNA was synthesized with Transcript one-step gDNA Removal and cDNA Synthesis supermix (Transgene Biotech Co., Ltd) and analyzed by real-time quantitative PCR with SYBR Premix Ex (RR420A, TaKaRa, Dalian, China). The reaction mixtures containing
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TaqTM
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SYBR Green were composed following the manufacturer’s protocol. Relative expression levels of the target genes were normalized to control gene 18s rRNA.
The sequences of the primers used for quantitative real time-PCR were as
follows: Fizz: forward-CCCTGCTGGGATGACTGCTA, reverse-TGCAAGTATCTCCACTCTGGATCT;
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ACCEPTED MANUSCRIPT PPAR-γ: forward- TTCGATCCGTAGAAGCCGTG, reverse- TTGGCCCTCTGAGATGAGGA; 18s rRNA: forward- CGGCTACCACATCCAAGGAA,
2.11 Conditioned medium preparation
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reverse- GCTGGAATTACCGCGGCT.
Macrophage polarization was obtained by culturing cells in DMEM
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medium supplemented with 10% FBS and 10 ng/ml IL4/IL13 or 5µM 4-HPR for
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three days. Then different polarized RAW264.7 cells were incubated in serum free medium for 24h, after which culture supernatants were collected as conditioned medium (CM). CM was centrifuged at 3000 rpm to separate out the debris and stored at -80
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2.12 Tube formation assay
HUVEC cells (3x104 cells per well) were seeded in 96-well plates that were filled with 50µl Matrigel and solidified in 37
. The cells were cultured in
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CM supplemented with 2.5% FBS for 6 hours. To observe the formation of
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tube-like structures, five optical fields (10x magnification) per well were randomly chosen and analyzed by a LEICA DMI 4000B microscope with Leica Application Suite software. 2.13 Western blot analysis After treatment with compounds for the indicated times, the macrophages were harvested. Cells were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM β-Sodium
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ACCEPTED MANUSCRIPT Glycerophosphate, 0.3% NP-40, 0.3% Triton X-100, 0.25% Leupeptin, 0.1% PMSF, 0.1% NaVO3). Cell lysates were centrifuged at 13,200 rpm for 30 min at 4
. Proteins were separated in 8% SDS-PAGE and blotted onto PVDF
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membranes, then incubated with primary antibodies followed by HRP-labelled secondary antibodies. The protein level was then detected using an ECL-plus kit and visualized on autoradiography film.
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2.14 Animals
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APCmin/+ mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The following primers were used for genotyping:
APC (wild type mice) Forward: 5’-GCCATCCCTTCACGTTAG-3’
APC
(for
both
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APC (APCmin/+ mice) Forward: 5’-TTCTGAGAAAGACAGAAGTTA-3’ wild
type
and
APCmin/+
mice)
Reverse:
5’-TTCCACTTTGGCATAAGGC-3’.
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Both genders were used in the experiments unless specifically indicated.
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The mice were housed under standard light/dark cycling and had access to food and water ad libitum. All the protocols were approved by the local ethics committee. To evaluate in vivo tumorigenesis, 6-week-old APCmin/+ mice were fed with a HFD (45% fat, Xietong organism, Nanjing, China). 2.15 TUNEL assay The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was used to evaluate the apoptotic response of tumor tissues
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ACCEPTED MANUSCRIPT in formalin-fixed tumor samples in paraffin blocks using the one-step TUNEL Apoptosis Assay kit (C1088, Beyotime, Nanjing, China) according to the manufacturer’s instructions.
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2.16 Histology and immunohistochemistry Tissues were fixed in 4% formaldehyde and embedded in paraffin. Then 3-5µm
sections
from
all
the
tissues
were
cut
and
stained
with
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hematoxylin/eosin (H&E) and reticulin. All the sections were reviewed by a
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pathologist. Immunohistochemistry was performed using an automated immunostainer with a DAB detection kit according to the company’s protocols for open procedures with slight modifications, using mouse monoclonal anti-CD206 (dilution 1:200, ab64693, Abcam, Cambridge, UK), anti-CD31
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(dilution 1:200, ab28364, Abcam, Cambridge, UK) and anti-Ki67 (dilution 1:50, sc-15402, Santa Cruz Biotechnology CA, USA). For antigen retrieval, sections were boiled in EDTA buffer (pH 9) for 30min prior to staining.
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2.17 Statistical analysis
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Values are presented as means ± SD. Two-tailed and unpaired Student’s t-test was used for statistical analysis, and differences were considered significant for p values less than 0.05.
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ACCEPTED MANUSCRIPT 3. Results 3.1 4-HPR efficiently inhibits M2 polarization of macrophages induced by IL-4/IL-13
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Because M2 TAMs mainly contribute to the poor progression of tumors, and 4-HPR has been shown to be a chemopreventive agent in clinical trials[25], it is interesting to assess whether 4-HPR could affect the M2 polarization of
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macrophages. IL-4/IL-13 induces a distinct functional and phenotypic
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response of macrophages often described as M2 macrophage polarization (compared to M1 polarization describing a macrophage phenotype in response to the Th1 cytokine IFN-γ or toll-like receptor (TLR) activation)[26, 27]. Therefore we first analyzed the impact of 4-HPR on IL-4/IL-13 induced M2-like
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polarization of macrophages in vitro. As expected, significant up-regulation of CD206 was observed when RAW264.7 cells were treated with either 10 ng/ml IL-4 or IL-13 for 48h (Figure 1A and 1B). Meanwhile, the expression of CD206
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induced by IL-4/IL-13 was greatly reduced by additionally treatment with 5µM
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4-HPR (from 28.61±4.08% to 11.48±2.41% and 33.29±2.55% to 14.21±2.69%, respectively) (Figure 1A and 1B). Since polarization of macrophages is accompanied by profound gene expression changes, we next measured the expression of specific M2 TAM marker genes, Fizz1 and PPAR-γ[28]. A significant decrease of these two genes was observed after co-administration of 5µM 4-HPR and IL-4/IL-13 compared with the IL-4/IL-13 only group (Figure 1C). The inhibition of M2 polarization is not caused by the 4-HPR-mediated
14
ACCEPTED MANUSCRIPT cytotoxicity (Figure S1A and S1B). To further validate the effect of 4-HPR on M2 polarization, we generated primary bone marrow derived macrophages (BMDMs) from mice and performed the same experiments. Again, IL-4/IL-13
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induced CD206 expression in BMDMs was totally blocked by 4-HPR (Figure 1D and 1E), and the decrease of M2 marker genes also confirmed the inhibition of M2 TAM polarization caused by 4-HPR (Figure 1F). Of note,
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4-HPR treatment did not block the M1 polarization in RAW264.7 cells caused
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by IFN-γ and lipopolysaccharides (LPS) (Figure S1C). Therefore, these results clearly suggest that 4-HPR can effectively and specifically inhibit the M2 polarization of macrophages in vitro.
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3.2 ROS is not involved in the 4-HPR-mediated inhibition of the M2 polarization of macrophages
We and other groups have proven that the generation of reactive oxygen
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species (ROS) mediates 4-HPR tumor cell cytotoxicity[15, 17, 29]. Thus, it is
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necessary to study whether ROS was also involved in 4-HPR-mediated inhibition of TAM M2 polarization. To address this issue, we first analyzed the ROS level of RAW264.7 cells treated with 4-HPR. As shown in Figure 2A, a significant generation of ROS was observed when the RAW264.7 cells were treated with 5µM 4-HPR. To investigate the relationship between the inhibition of M2 polarization and ROS, we applied the ROS scavengers N-acetylcysteine (NAC) and glutathione (GSH) to remove intracellular ROS generated in
15
ACCEPTED MANUSCRIPT response to 4-HPR treatment. Intriguingly, the NAC and GSH promoted the inhibition ratio of M2 polarization of macrophages treated with 4-HPR, which indicated that ROS might promote rather than inhibit the M2 polarization
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(Figure 2B). In order to confirm the general role of ROS in M2 polarization, we next used H2O2 to generate ROS and test the impact of H2O2 on the M2 polarization. As shown in Figure 2C and 2D, H2O2 alone or together with
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IL-4/IL-13 could induce or promote CD206 expression. Moreover, the increase
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of CD206 expression in macrophages with IL-4/IL-13 and H2O2 could be reversed by NAC and GSH (Figure 2E). Taken together, these results suggest that 4-HPR mediated the inhibition of TAMs M2 polarization in an
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ROS-independent manner.
3.3 The STAT6 signaling pathway participates in 4-HPR-mediated inhibition of M2 polarization
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The classical pathway for IL-4-induced TAM polarization involves the
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activation of the STAT6 signaling pathway[30]. Once STAT6 is activated, it can bind directly to the promoter of certain downstream genes such as Fizz1 and transcriptionally activates these genes[31]. As 4-HPR inhibited M2 polarization specific gene Fizz1 gene expression, we next assessed whether STAT6 participated in 4-HPR-mediated inhibition of M2 macrophage polarization. In general, STAT6 is activated rapidly upon cytokine receptor binding. IL-4 can sustain the phosphorylation level of STAT6 during the polarization process.
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ACCEPTED MANUSCRIPT Thus we first investigated the phosphorylation of STAT6 after treatment with IL-4/IL-13 with or without 4-HPR. As shown in Figure 3A and 3C, in RAW264.7 and BMDM cells, the phosphorylation status of STAT6 was increased after
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treating cells with IL-4/IL-13 for 2h. Meanwhile the increase of phosphorylated STAT6 was dramatically inhibited by co-treatment with 4-HPR, which suggests that STAT6 might participate in 4-HPR-mediated inhibition of TAMs M2
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polarization (Figure 3B and 3D). To further confirm this result, we applied the
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siRNA to specifically knockdown the intracellular protein expression of STAT6, and then measured the change of the inhibition of M2 polarization caused by 4-HPR. As shown in Figure 3E, two siRNAs against STAT6 remarkably decreased the endogenous STAT6 expression in cells. Moreover, the STAT6
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silencing by siRNA also significantly reduced the M2-like polarization induced by IL-4/IL-13 as expected. Of note, the blockade of STAT6 expression did not further increase the inhibitory ratio of CD206 caused by 4-HPR (Figure 3F),
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which suggested that inhibition of the phosphorylation of STAT6 significantly
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affected 4-HPR-inhibited M2-like polarization. Therefore, our data support the hypothesis that the STAT6 signaling pathway participates in 4-HPR-mediated inhibition of M2 polarization.
3.4
4-HPR
abrogates
the
angiogenesis
phenotype
of
M2-like
macrophages The next key question is whether 4-HPR-mediated inhibition of M2
17
ACCEPTED MANUSCRIPT polarization could result in the inability of tumors [32]. In order to address this issue, we first induced the inhibitory effect of 4-HPR directly on three colorectal cell lines, HCT116, SW620 and Colo 205, to rule out the possibility that
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inhibition of M2 polarization is dispensable for 4-HPR-driven chemoprevention. As shown in Figure S2A, the proliferation of cells treated with 4-HPR was not significantly inhibited compared to those treated with 5-Fluorouracil (5-Fu),
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which is a first-line treatment for colorectal cancer. In addition, no significant
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apoptosis was observed after the treatment with 4-HPR for 72h compared with the treatment with 5-Fu (Figure S2B).
Next, we focused on the two well-known phenotypes of M2 TAMs proliferation promotion and angiogenesis promotion[33, 34]. Macrophages
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were treated with IL-4/IL-13, 4-HPR, or both for 72h, and the culture medium was replaced with fresh medium without serum. Then, 24 h later the supernatant medium was collected (referred to as CM). To evaluate the impact
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of CM on tumor cell survival, the colorectal cell lines HCT116, SW620, and
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SW480 were treated with the CM for 72h and cell proliferation and apoptosis were further analyzed. Surprisingly, no significant difference was found in the IL-4 combined 4-HPR group (vs. the IL-4 group), or the IL-13 combined 4-HPR group
(vs.
the
IL-13
group)
(Figure
4A
and
4B).
For
the
angiogenesis-promoting effect, we evaluated the tube formation of HUVEC cells treated with CM. HUVEC cells were seeded in a 96-well plate pre-coated with Matrigel and formed capillary-like structures in the presence of different
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ACCEPTED MANUSCRIPT supernatants of macrophages. As shown in Figure 4C, the network of tube-like structures in the IL-4/IL-13 group was more extensive than those in the IL-4/IL-13 combined with 4-HPR groups (yellow arrow indicated, Figure 4D).
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Together with the inhibition of M2 polarization we described previously, these data suggest that the subcytotoxic concentration of 4-HPR could abrogate the
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physiological angiogenesis-promoting function of M2 TAMs.
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3.5 4-HPR prevents the colorectal cancer formation in vivo by targeting macrophages
In order to validate our result with an in vivo approach, we then tested whether 4-HPR blocks tumor formation as well as inhibits the occurrence of
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M2 TAMs in APCmin/+ transgenic mice, which are highly susceptible to spontaneous intestinal adenoma formation[35]. Considering that monocytes are constantly produced from bone marrow, we hypothesized that continuous
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4-HPR treatment is necessary to block the differentiation of monocytes into
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TAMs. Six-week-old APCmin/+ mice were maintained on 4-HPR (20mg/kg) for 14 weeks together with a high fat diet. Whole intestines from these mice were collected, and histopathological examination of H&E-stained sections of the intestines was completed in all the mice (Figure 5A). Of note, the number of intestinal tumors was significantly reduced in the 4-HPR treated group as compared to the control group, from 14.33+7.73 to 6.11+7.00 (p<0.01) (Figure 5B and 5C), without affecting the body weight of the mice (Figure 5D).
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ACCEPTED MANUSCRIPT To explore whether TAMs played a critical role in 4-HPR-inhibited tumorigenesis, we then tested if 4-HPR altered TAM polarization in tumor tissues. Immunohistochemistry analysis showed that 4-HPR inhibited TAM M2
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polarization, as less CD206 in 4-HPR treated tumors was detected in the tumor region (Figure 6A). Similar results are shown in Figure 6B, which were determined using log IOD in Image-Pro Plus 6.0, indicating that the expression
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of CD206 was correlated with the tumorigenesis of APCmin/+ mice. Since the
support
the
fact
that
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status of tumor vessels contributes to tumorigenesis and our in vitro data 4-HPR
abolished
the
physiological
angiogenesis-promoting function of M2 TAMs, we tested if 4-HPR affected vessel density in mice. As shown in Figure 6G and 6H, CD31 staining revealed
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that tumor vessel density was dramatically decreased in tumors treated with 4-HPR. Moreover, we also investigated whether 4-HPR effected on the tumor growth and apoptosis. As shown in Figure 6C and 6E, 4-HPR did not induce
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colorectal tumor cell death nor inhibit the cancer cell growth.
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Collectively, these observations suggest that the M2-like phenotype plays a positive role in regulating tumorigenesis in colorectal cancer; nevertheless, 4-HPR could antagonize the tumorigenesis of colorectal cancer induced by M2-like macrophages.
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ACCEPTED MANUSCRIPT 4.Discussion Alhough 4-HPR has received great attention as a chemopreventive agent, based on chemoprevention clinical trials, the underlying mechanism of its
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chemopreventive effect is still poorly understood. Using RAW264.7, we demonstrated the remarkable inhibition of M2 macrophage polarization by the administration of 4-HPR. It should be emphasized that the effective
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concentration of 4-HPR for M2 macrophage polarization inhibition is lower than
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the concentration used for tumor cytotoxicity, which might be a more reasonable mechanism to explain its chemopreventive activity in the clinic. Importantly, although there are several clinical trials of 4-HPR as a cancer chemoprevention agent, none have identified the potential biomarkers in the
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inclusion criteria. Specifically, our finding that M2 macrophages are dramatically decreased in APCmin/+ transgenic mice further suggests that the analysis of an M2 polarization marker in tumor biopsy sections may identify the
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biomarker of the chemopreventive effect of 4-HPR.
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We and other groups have previously reported that 4-HPR inhibited tumor growth and metastasis in animals and functioned as an apoptosis inducer mostly through the production of ROS and mitochondrial disruption[16, 17, 36]. When we treat RAW264.7 cells with H2O2 alone or together with IL-4/IL-13, it could induce or promote CD206 expression, suggesting the positive role of ROS in M2 polarization. These results are consistent with a previous report that ROS elimination by butylated hydroxyanisole and other ROS inhibitors
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ACCEPTED MANUSCRIPT blocks M2 macrophage polarization[37]. However, according to our finding ROS are not involved in 4-HPR-induced inhibition of M2 macrophages. Instead, we found that 4-HPR-induced inhibition of M2 macrophages correlates with
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decreased phosphorylation status of STAT6. Although the underlying mechanism of 4-HPR-triggered inhibition of STAT6 is unknown, based on the fact that 1) ceramide could active several signaling pathways including the
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JAK/STAT axis[38], and 2) recent in vitro data indicates that 4-HPR disrupts
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the biosynthesis of ceramides through the inhibition of dihydroceramide desaturase (Des1)[39], we propose that the inhibition of ceramide synthesis might mediate 4-HPR-triggered inhibition of STAT6.
Two well-known phenotypes of M2 TAMs are proliferation-promotion and
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angiogenesis-promotion[40, 41]. However, in the current study, we did not observe the proliferation promoting effect of M2 macrophages on three colon cancer cell lines, which might explain why we failed to detect a difference in
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colon cancer cell proliferation in the presence of conditioned media derived
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from 4-HPR-treated M2 macrophages. Alternatively, both in vitro and in vivo data suggest that 4-HPR does abrogate the angiogenesis phenotype of M2-like macrophages. Interestingly, previous reports also have suggested that the chemopreventive effects of 4-HPR in early intervention protocols are likely due to its anti-angiogenesis properties. Macrophages represent 10%-20% of all mononuclear cells found in the intestinal lamina propria, making the intestine the largest reservoir of
22
ACCEPTED MANUSCRIPT macrophages in humans[42, 43]. The identification of cancer-induced macrophages differentiation characteristics is helpful for understanding the correlation between TAMs and colorectal cancer[44, 45]. The immunological
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role of TAMs in the prognosis of CRC patients has been debated for the past few decades[46, 47]. The inconsistency among these studies is likely due to the cancer characteristics, the preparation of the macrophages studied, and
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the definition of M1/M2 macropahges. Understanding the early events in
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colorectal carcinogenesis is still incomplete. Accumulating evidence has indicated that inflammatory responses are important for cancer development. Epidemiological studies have shown that regular use of non-steroidal anti-inflammatory drugs reduces the risk of colorectal cancer development[48].
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In the tumor microenvironment, tumor-associated macrophages express pro-inflammatory cytokines, including CCL2, IL-4, IL-13, etc. Recent studies have uncovered the role of such an inflammatory network in the promotion of
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gastrointestinal tumor development[13, 49]. Genetically engineered and
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chemically induced mouse tumor models, which mimic sporadic or inflammation-associated tumorigenesis were used in these studies. In our study, we first demonstrated that macrophages might be involved in colorectal cancer chemoprevention. Although several studies revealed that TAMs are a biomarker of colorectal cancer prognosis, none of them ever clearly investigated the relationship between the inhibition of TAMs and tumorigenesis of colorectal cancer[50].
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ACCEPTED MANUSCRIPT In summary, we identify 4-HPR as selectively inhibiting M2 macrophage polarization through the inhibition of phosphorylation of STAT6, and in turn preventing the tumorigenesis of colorectal cancer. Macrophage modulation
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might therefore contribute to the chemopreventive effect of 4-HPR. Incomplete inhibition of macrophage activation during 4-HPR treatment indicates that more efficient strategies are needed to achieve a robust modulation of
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macrophages in the tumor environment and further improve tissue response.
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Our data suggest that specifically addressing the STAT6 signaling pathway is a promising approach to modulate the CRC environment.
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ACCEPTED MANUSCRIPT Figure Legends Figure 1: 4-HPR inhibits M2 polarization of macrophages induced by IL-4/IL-13 (A and B) RAW264.7 cells were exposed to IL-4/IL-13 (10 ng/ml)
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Figure 2: 4-HPR inhibits M2 polarization of macrophages in a ROSindependent manner. (A) RAW 264.7 cells were treated with 4-HPR for the indicated time and then harvested for DCFH-DA analysis by flow cytometry. (B)
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ACCEPTED MANUSCRIPT Figure 3: The canonical STAT6 pathway was involved in 4-HPR-induced inhibition of M2-like polarization of macrophages. (A and C) RAW264.7 cells and BMDMs were treated with IL-4/IL-13 with or without 4-HPR (5 µM) for
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and Methods. Proteins were detected by western blot analysis using specific antibodies. (F) RAW 264.7 cells were harvested and analyzed for CD206 (n=3).
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Figure 4: 4-HPR abrogates the angiogenesis phenotype of M2-like macrophages. (A)Proliferation of colorectal cancer cells (HCT116, SW620, and SW480) in the presence of conditioned media derived from 4-HPR-treated
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Figure 5: 4-HPR prevented colorectal cancer in vivo. (A) Schematic
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Figure 6: 4-HPR prevented colorectal cancer in vivo by targeting (A)
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Highlights 4-HPR could significantly suppress M2-like polarization of macrophages. The inhibition of phosphorylation of STAT6, rather than the generation of oxidative stress, is involved in the 4-HPR-driven inhibition of M2 polarization. The inhibition of M2 polarization in involved in 4-HPR-mediated chemoprevention