Background
Although the incidence of gastric cancer (GC) has greatly reduced in developed countries, it remains the third leading cause of cancer-related deaths worldwide [
1], and one of the most common cancers in Asia. In China, GC is the third leading cause of cancer-related deaths, and the proportion of advanced GC is about 70%. Although targeted drugs prolong the survival of patients with advanced GC by more than a year, the 5-year survival rate is less than 20% [
2,
3]. Therefore, new treatment strategies need to be developed.
Cancer immunotherapy has shown major advancements during the past few years. Immune checkpoints such as cytotoxic T-lymphocyte-associated antigen (CTLA-4) and programmed death-ligand 1 (PD-L1) can suppress the activity of T-lymphocytes, which could recognize and eliminate cancer antigens [
4]. Therefore, blocking programmed death- 1 (PD-1) is now an attractive medical approach to enhance anti-tumor immunity [
5‐
7]. In 2014, the PD-1 monoclonal antibody pembrolizumab targeting the PD-L1/PD-1 signaling pathway, had shown significant clinical effect in patients with high PD-L1 expression in advanced melanoma and non-small cell lung cancer. Moreover, it was approved by the US FDA as the first-line treatment for advanced melanoma in 2015 [
8]. Recently, the clinical trial KEYNOTE-012 showed a good clinical effect of pembrolizumab in PD-L1-positive advanced GC [
9], which suggested that PD-L1-positive GC patients might benefit from blocking the PD-L1/PD-1 signaling pathway. Therefore, researchers and physicians have paid increasing attention to cancer immunotherapy, and the molecular target therapy of immunological checkpoints has brought new hope for solid tumors, with PD-L1 being a promising one.
Majority of the recent studies have shown that PD-L1 expression is regulated by intrinsic and extrinsic mechanisms in cancer cells. Intrinsic immunologic tolerance associates carcinogenesis with PD-L1 expression, such as overexpression of ALK and Ki-67, activation of Kras, and inactivation of PTEN and Lkb1 [
10‐
14]. Additionally, several reports have suggested that extrinsic immunologic tolerance plays an important role in the regulation of PD-L1. Various cytokines play an important regulatory role in tumor microenvironment. Interferon-γ (IFN-γ) secreted by activated immune cells is not only an important anti-tumor factor but also a central extrinsic factor that induces upregulation of PD-L1 in tumor cells [
15‐
17]. Recently, cytokines, such as interleukin-27 (IL-27), IL-17, tumor necrosis factor-α (TNF-α) [
18,
19], etc., have been found to regulate PD-L1. Therefore, it is important to elucidate the regulatory mechanism between cytokines and PD-L1.
In the present study, bioinformatics was used to explore the PD-L1-related genes in GC and propose a hypothesis. In vitro experiments were conducted to confirm the hypothesis, which clarified that CXCL9/10/11-CXCR3 upregulated PD-L1 expression by activating the STAT and PI3K-Akt pathways in SGC7901 and MKN74 GC cell lines. These findings suggest a novel mechanism regulating the immune-evading factor PD-L1.
Methods
GSEA analysis
RNASEQV2 dataset was obtained from the Cancer Genome Atlas (TCGA) (
https://gdc-portal.nci.nih.gov/). Another two mRNA expression profiles (GSE15459 and GSE62254) were downloaded from Gene Expression Omnibus (GEO) (
http://www.ncbi.nlm.nih.gov/geo/). Above two datasets were acquired using Affymetrix Human Genome U133 Plus 2.0 Array. TCGA dataset included surgically resected 375 GC samples with complete data. Dataset GSE15459 included surgically resected 192 GC samples with complete data from the Singapore patient cohort [
20]. Dataset GSE62254 contained surgically resected 300 GC samples with complete data from the Korean patient cohort [
21].
The TCGA data was preprocessed according to the Bioconductor/TCGABiolinks function package flow. The gene expression profile data were extracted using the Bioconductor/GEOquery function package. Gene CD274 corresponds to two probes, “223834_at” and “227458_at”, and subsequent analysis was performed on the above two probes. The expression of CD274 in the samples was sorted from high to low, with the sample whose signal above the median value as the high expression group and below the median as the low expression group.
The GSE62254, GSE15459 and TCGA-STAD-RNASEQV2 data were substituted into GSEA2.0.14 to analyze the effect of high and low expression level of CD274 on various pathways of biological pathways, and the reference gene set was set to the c2.cp.kegg.v5.1.symbols.gmt gene set in the MsigDB database of the GSEA website. The analysis was repeated 1000 times for each one according to the default weighted enrichment statistic method. In the GSEA, based on the condition of P < 0.05 and false discovery rates (FDR) < 0.25, the first 20 pathways were ranked as the “significant enrichment genes” according to the FDR value from low to high,if P = 0 and FDR = 0, the pathways should be included. The software R 3.2.3 was used to perform all the statistical analyses.
Functional enrichment analysis
Based on TCGA dataset, the correlation coefficient r between CD274 and the expression of each gene was calculated by Spearman correlation analysis. Based on the condition of
r > 0.6, genes were selected for further analysis. The database for annotation, visualization and integrated discovery (DAVID) was used to analyze list of genes derived from high-throughput genomic experiments. DAVID online tool [
22] for Gene functional classificationwas used to perform the enrichment analysis of the biological processes of CD274 related genes in order to identify the enriched pathways and genes [
23].
Reagents and antibodies
CXCL9/10/11 were purchased from Pepro Tech (USA). The PD-L1 antibody was purchased from Cell Signalling Technology (USA). The CXCR3 antibody was purchased from ABCAM (USA). The CXCR3 antagonist AMG487 was purchased from Tocris (USA). The PI3K/AKT inhibitor LY294002 and the STAT3 inhibitor STATTIC were obtained from Sigma (St. Louis, MO, USA). The anti-STAT3, anti-pSTAT3, anti-Akt, anti-pAkt, anti-ERK, anti-pERK, and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (USA).
Cells and cell culture
Human gastric cell lines SGC-7901 and MKN74 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (China). Cells were grown in RPMI 1640 (Rosewell Park Memorial Institute) containing with 10% fetal bovine serum and 1% penicillin–streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C [
24].
Western blot analysis
Cells were seeded at 2 × 10
5 per well in 6-well plates and incubated overnight; Cells were treated with CXCL9/10/11 (100 ng/ml) for indicated times. Cells were lysed in lysis buffer. The method of Western blot was discussed in our previous study [
25].
Small interfering RNA transfections
The siRNA sequences were from View solid biotechnology co., LTD (Beijing, China). The AKT siRNA sequence was 50-CUCACAGCCCUGAAGUACUtt-30. The siRNAs were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) per the manufacturer’s instructions.
Patients and tissue specimens
The files of 92 patients who underwent surgical resection of gastric cancer between 2006 and 2011 at the First Hospital of China Medical University, were included in our study. Patient information included age, gender, TNM stage, histopathologic type and metastases. All patients were not treated with any chemotherapy. This study was approved by the Human Ethics Review Committee of China Medical University; informed consent was obtained from all patients.
Immunohistochemistry and evaluation
Immunohistochemical staining was performed using a streptavidin-peroxidase procedure. CXCR3 and PD-L1 expression were investigated using antibodies against CXCR3 (Abcam, USA) and PD-L1 (Cell Signalling Technology, USA). All other reagents were from Sigma. The specificity of all antibodies was confirmed by western blotting.
PD-L1 and CXCR3 were detected by immunohistochemistry (IHC) as described previously. Staining was graded into four categories: 0 (staining equal to or less than 5%), 1+ (staining of 6–25%), 2+ (staining of 26–50%), 3+ (staining more than 50%). Tissues with scores of 2+, or 3+ were considered to be positive [
26]. All histological and IHC slides were evaluated by a certified surgical pathologist in our department.
Statistical analysis
All statistical tests were performed using SPSS (Statistical Package for the Social Sciences) 17.0 computer software. Differences between two groups were evaluated by Student’s t-test. A P-value of 0.05 or less was considered to indicate a statistically significant difference. Spearman correlation analysis was used to determine the correlation of PD-L1 and CXCR3 expression.
Discussion
PD-L1 expression is regulated by intrinsic and extrinsic pathways, and could be induced by hypoxia, toll-like receptor (TLR), gene mutations and cytokines. IFN-γ of the cytokine family is a key factor triggering PD-L1 induction in tumor cells. However, the specific mechanisms underlying PD-L1 expression remain largely unknown. Stimulation with IFN-γ, TLR ligands and LPS via MyD88, TRAF6, MEK, STAT1, NF-κB and PI3K-dependent pathways increased PD-L1 expression in different types of cells, such as inflammatory cells, fibroblasts and cancer cells [
27‐
30]. Additionally, IL-12, IL-27, IL-17 and TNF-α regulated PD-L1 expression in inflammatory cells and cancer cells by altering NF-κB, STAT1 and ERK1/2 signaling [
18,
19,
31]. However, our study showed that PD-L1 expression was enhanced via chemokine subfamily CXCL9/10/11-CXCR3 in a STAT3 and Akt-dependent manner in GC cells, suggesting that cytokines may induce PD-L1 expression through different STAT pathways. Our data do not support a role for ERK in inducing PD-L1 expression, but it seems likely that IFN-γ signaling is mediated through different pathways depending on the cell types involved.
CXCL9/MIG, CXCL10/IP10 and CXCL11/ITAC are members of the ELR-negative CXC chemokine subfamily, also known as IFN-γ-induced monocyte cytokines, IFN-γ-inducible protein 10 and IFN-γ-inducible T cell α chemokine [
32‐
34]. CXCR3, the co-receptor of CXCL9/10/11, belongs to the seven-transmembrane G protein-coupled receptors (GPCRs) and is mainly expressed on activated T cells, NK cells, mast cells and dendritic cells. CXCR3 is reported to play a dual role in immunity and cancer. The CXCL9/10/11-CXCR3 signaling pathway in tumor microenvironment mainly facilitated the chemotactic movement of CXCR3 activated immune cells to the tumor site for anti-tumor immunity [
35]. While CXCR3 expression was also detected in many cancers, such as breast cancer, malignant melanoma, renal cancer and colon cancer [
36‐
39]. Some studies suggested that IFN-γ induced ELR-negative CXC chemokine expression, which can activate downstream MAPKs, PI3K-Akt and STAT by binding to their receptor CXCR3, and thereby promote tumor progression and metastasis. Meanwhile, IFN-γ as an immune factor plays an important role in the regulation of PD-L1. Interestingly, our study found that CXCR3 and PD-L1 were expressed in GC cells and tissues, and CXCL9/10/11-CXCR3 upregulated PD-L1 expression by activating STAT and PI3K-Akt pathways in GC through both data analysis and in vitro experiments, which meant that CXCR3 could play anti-tumor and pro-tumor roles in different types of cells.
Conclusions
In summary, our findings suggest a regulatory mechanism of PD-L1 through data analysis, in vitro and vivo experiments, which is an important factor of immune evasion in GC cells, and CXCL9/10/11-CXCR3 could regulate PD-L1 expression through STAT and PI3K-Akt signaling pathways in GC cells. Recently, molecular targeted therapy has created hope for advanced GC patients. Checkpoint blockers such as PD-L1 offer novel immunotherapy options for cancer patients. Based on our findings, CXCR3 could be a potential target in GC therapy. Further studies are required to confirm this conjecture.
Acknowledgements
We want to thank Dr. Kezuo Hou for his help with the experimental reagents and materials.
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