Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Abstract Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The ApcMin/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, ApcMin/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of ApcMin/+ WT and ApcMin/+ TLR4−/− mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of ApcMin/+ TLR4−/− mice compared with ApcMin/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.


| INTRODUC TI ON
Colorectal cancer (CRC) has become the second leading cause of cancer-related death in developed countries. The transitional treatments include surgery, combined chemotherapy and radiation therapy. 1 Currently, the use of targeted therapies is limited to patients with stage IV metastatic colon cancer. 2 Thus, the identification of new targets for CRC therapy is needed.
Previous studies have reported that the Apc/Wnt pathway is an attractive target for the treatment of CRC. 3 The adenomatous polyposis coli (Apc) tumour suppressor is mutated in 80%-90% of human colorectal tumours. 4 Apc, along with axin and glycogen synthase kinase 3b (GSK3b), assembles a protein complex that targets β-catenin for degradation, which is regulated by Wnt signalling. 5 This is the main mechanism of Apc mutation leading to tumorigenesis, as the resulting increased availability of β-catenin causes changes in the transcriptional process to decrease the amount of cell differentiation. 6 TLRs are sensors for pathogen-associated molecular patterns (PAMPs) and play important roles in initializing inflammation. 7 Toll-like receptor 4 (TLR4) is the main mediator of responses to lipopolysaccharide (LPS) both in vitro and in vivo. 8 It has been reported that inflammatory responses contribute to intestinal carcinogenesis through multiple mechanisms, for example TLR4 signalling activates NF-κB through the MYD88 pathway, [9][10][11] and it is well known that patients with inflammatory bowel disease are at higher risk of CRC. 12 TLR4/MYD88 signalling contributes to CRC tumorigenesis not only in colitis-associated cancer, but also in sporadic CRC. 13 Disorders of intestinal microflora can also lead to intestinal carcinogenesis. Chen

et al reported that
Fusobacterium nucleatum activates beta-catenin signalling in CRC via a TLR4/P-PAK1 cascade. 14 However, it has been reported that TLR4-deficient C3H/HeJ mice developed more tumours relative to TLR4-normal C3H/HeN mice. 15 This suggests that TLR4 may also have a role in inhibiting tumorigenesis. It is therefore evident that the specific regulation mechanism of TLR4 signalling that promotes or inhibits tumorigenesis is unclear. In the present study, Apc Min/+ (multiple intestinal neoplasia) mice were generated on TLR4-sufficient and TLR4-deficient backgrounds to explore the role of this pathway in spontaneous intestinal tumorigenesis.
Apc Min/+ mice carry a single mutant Apc allele and develop  benign adenomas in the gut by 4-5 months of age. 3 In this study, high-throughput sequencing was used to screen out the production of TLR4 signalling downstream genes and validate them in a variety of ways. These data may provide an insight into the role of TLR4 in intestinal tumorigenesis.

| Mice
Adult WT mice (C57BL/6 wild type) were purchased from the Chinese Academy of Science (Shanghai, China). TLR4 −/− , Apc Min/+ WT and Apc Min/+ TLR4 −/− mice were provided by the Model Animal Research Center of Nanjing University (Nanjing, China).

| Analysis of peripheral blood
To explore the peripheral blood conditions, the mice were eyebled using heparin-coated capillary tubes to take blood while the mice were under anaesthesia. The blood was transferred to Eppendorf (EP) tubes with K2-EDTA and inverted multiple times. A small animal blood cell counter was used to measure blood cells.

| Measurement of bone marrow haematopoietic precursor cells
Femur bone marrow was taken from each group of mice to make bone marrow nucleated cell suspensions. After the bone marrow nucleated cell suspensions were diluted to the appropriate concentration, the corresponding fluorescently labelled antibody was added in a ratio of 1:100 (GMP: Lin − c-Kit + FcγRII/III hi CD34 hi ; CMP:

| Histological study
Gut tissues were dehydrated in an ascending grade of ethanol, then cleared and embedded in paraffin wax. Serial sections of 2-7 microns thick were obtained using a rotatory microtome. The deparaffinized sections were stained routinely using the H & E staining method.
Ki67 kits were applied to assess the proliferative capacity of intestinal epithelial crypt cells. 20 TUNEL kits were applied to assess the apoptosis of intestinal epithelial crypt cells. 21 Photomicrographs of the desired sections were obtained using a digital research photographic microscope (Thermo Corporation, USA).

| Quantitative real-time PCR (RT-qPCR)
Total RNAs from tissue samples were isolated using an RNA isolation and purification reagent kit provided by GenePharma Corporation were normalized for the number of cytokines per mg of total protein in the supernatant. 24 In the experiments of IL-6 and GM-CSF mediated by TLR4, the mice were abdominally injected with 1 mg/ kg bodyweight LPS, and the CT-26 cells were treated with 1 ng/ mL of LPS 24 hours before measuring the IL6 and GM-CSF with the ELISA kit.

| Flow multifactor detection assay
The mean fluorescence intensity of the cytokines in the serum of each group was measured by a multifactor detection reagent kit 25 (Dakewe Biotech Corporation, Shenzhen, China), and flow cytometry (Beckman Coulter, USA) was utilized for testing.

| MTT assay
Relative cell viability was analysed using tetrazolium salt 3-(4, The medium was removed, and the cells were solubilized in 150 μl of dimethyl sulphoxide before colorimetric analysis was performed (wavelength: 490 nm). One plate was analysed immediately after the cells adhered (approximately 4 hours after plating), and the remaining plates were analysed every day for the next 3 days. 26

| Apoptosis analysis
Cell apoptosis was analysed using apoptosis detection kits 27 (TransGen Biotech, Beijing, China). The CT-26 cells were treated with 100 ng/mL of LPS, IL6, GM-CSF, IL11, CD40, TNF, CCL3, MMP9 and S100A8/A9 for 24 hours, and were then exposed to 6 Gy radiation irradiation or treatment with the chemotherapy drug paclitaxel. The SW1116 cells were treated with 100 ng/mL of LPS, IL6, GM-CSF and IL11 for 24 hours, and were then exposed to 6 Gy radiation irradiation or treatment with the chemotherapy drug paclitaxel. After

| Amounts of MDSC analysis in spleen and bone marrow of mice
Femur bone marrow was taken from each group of mice to make bone marrow nucleated cell suspensions (BMCs). The spleen taken from each group of mice was mashed. After the BMCs and spleen suspensions were diluted to the appropriate concentration, the corresponding fluorescently labelled antibody CD11b/Gr 1 (eBioscience Corporation, USA) was added. The suspensions were mixed well and incubated for 20 min at room temperature in the dark; PBS was then added to mix and dilute, and flow cytometry (Beckman Coulter, USA) was used for testing. Statistical analysis generated a P-value and statistic value for each analysis; P < .05 was considered a statistically significant difference.

| IL-6 and GM-CSF were mediated by TLR4 in WT or Apc Min/+ mice in protein levels
To explore the expression levels of IL6 and GM-CSF regulated by TLR4 signalling, three levels of experimental studies were conducted: in the serum and colons of the mice, and in the colon cancer  Figure 6B).
These data suggest that the expressions of IL6 and GM-CSF were regulated by TLR4 signalling in protein levels.

| Functional study of down-regulation factors such as IL6 and GM-CSF in intestinal cancer cell lines
CT-26 cells belong to the mouse intestinal cancer cell line. CT-26 cells were treated with LPS (TLR4 agonist), IL-6, IL-11, GM-CSF, CD40, TNF, CCL3, MMP9, S100A8 and S100A9, and cell viability F I G U R E 6 IL-6 and GM-CSF were mediated by TLR4 in WT or Apc Min/+ mice. (A) The left panels are, respectively, the expression levels of IL6 and GM-CSF in the serum of WT with or without LPS treatment (1 mg/kg bodyweight), and Apc Min/+ mice with or without LPS treatment (1 mg/kg bodyweight). The middle panels are the expression levels of IL6 and GM-CSF in the colons of Apc Min/+ mice with or without LPS treatment (1 mg/kg bodyweight). The right panels are the expression levels of IL6 and GM-CSF in CT-26 cells with or without LPS treatment (1 ng/mL). (B) The left panels are, respectively, the expression levels of IL6 and GM-CSF in the serum of Apc Min/+ mice with or without LPS treatment (1 mg/kg bodyweight), and Apc Min/+ TLR4 −/− mice with or without LPS treatment (1 mg/kg bodyweight). The middle panels are, respectively, the expression levels of IL6 and GM-CSF in the serum of WT mice with or without LPS treatment (1 mg/kg bodyweight), and TLR4 −/− mice with or without LPS treatment (1 mg/kg bodyweight). The right panels are, respectively, the expression levels of IL6 and GM-CSF in the colons of Apc Min/+ mice with or without LPS treatment (1 mg/kg bodyweight), and Apc Min/+ TLR4 −/− mice with or without LPS treatment (1 mg/kg bodyweight). *P < .05, **P < .01, ***P < .001; NS: No significant difference detected. All data were detected by ELISA assay was detected after 24 h using MTT assay. The results showed that, except for TNF, MMP9 and CD40, the other cytokines ef-

| D ISCUSS I ON
Vogelstein et al 29 held that patients with germline mutations of Apc did not necessarily develop CRC, along with additional risk factors, such as inflammation induction leading to tumour development. TLR4 pathway-mediated immune response is an important mechanism that aggravates intestinal injury in mouse ulcerative colitis models. 30 In the present study, Apc Min/+ intestinal adenoma mice were generated on TLR4-sufficient and TLR4-deficient backgrounds to investigate the carcinogenic effect of TLR4 by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc Min/+ WT and Apc Min/+ TLR4 mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. To study the downstream genes involved in the promotion of intestinal tumorigenesis by TLR4 signalling, high-throughput RNA sequencing was utilized to screen for dysregulated genes in gut tumours between Apc Min/+ WT and Apc Min/+ TLR4 mice. After a series of validation experiments for the concerned genes, it was found that IL6, GM-CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in the gut tumours of Apc Min/+ TLR4 −/− mice compared with Apc Min/+ WT mice. Combined with the KEGG enrichment data, it can be determined that TLR4 might promote intestinal tumorigenesis by activating cytokine-cytokine receptor interaction and pathways in cancer signalling pathways. In the functional study of core downregulation factors, it was found that IL6, GM-CSF, IL11, CCL3 and S100A8/9 increased the viability and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment. The effects of IL6 and GM-CSF were the most obvious.
The tumour-promoting effect of inflammation is now widely recognized and better understood. 31 The NF-κB-IL6-Stat3 cascade is an important inflammatory regulator of the proliferation of tumourinitiating intestinal epithelial cells. 32 Baltgalvis et al 33  TLR4 was the upstream molecule of IL6 that promoted tumour development, and it has been reported that TLR4 and NF-κB signalling are required to control the regulation of the IL-6 mRNA stabilizing molecule Arid5a. 34 GM-CSF was initially classified as a haematopoietic growth factor. In the inflammatory process, GM-CSF serves as a communication conduit between tissue-invading lymphocytes and myeloid cells, and GM-CSF-activated phagocytes are well equipped to cause tissue damage. 35 Chen et al 36 reported that GM-CSFtreated cancer cells exhibited an enhanced ability of motility both in vitro and in vivo, and that chronic exposure of intestinal cancer cells to GM-CSF led to the occurrence of intestinal epithelium to mesenchymal transition (EMT). Ripk3 signalling promotes intestinal tumours by up-regulating cytokines IL23 and IL1β, which are required for expanding IL-17-producing T cells through I-MDSCs, a distinct MDSC subset that is dependent on GM-CSF. 37 Ripk3 activity is mediated by TLR4. 38 We ever used CD11b/Gr 1 double staining to detect the expression of MDSC in spleen and bone marrow of WT, Apc Min/+ , Apc Min/+ TLR4 −/− mice by flow cytometry.
The results suggested that the expression level of MDSC in spleen and bone marrow of Apc Min/+ mice was higher than that in WT or Apc Min/+ TLR4 −/− mice ( Figure S4A

CO N FLI C T S O F I NTE R E S T
The authors have no competing interests to declare.