IRF-2 inhibits cancer proliferation by promoting AMER-1 transcription in human gastric cancer

Tumor specimens were obtained from 72 patients with gastric adenocarcinoma (GAC) who underwent curative resection at Zhongshan Hospital of Fudan University between 2011 and 2014. The inclusion and exclusion criteria were as follows: (a) having a distinctive pathologic diagnosis of GAC. (b) Curative gastric surgical treatment with complete resection of all cancer nodule, and histological examination revealing no tumor cells on the cut surface. (c) Complete follow-up data until June 2017. (d) Suitable formalin-fixed and paraffin-embedded tissues. (e) Patients who agreed to participate in the study and signed informed consent. The GAC diagnosis was based on WHO criteria, and the tumor stage was classified according to the 7th edition of the tumor-node-metastasis (TNM) classification by the Union for International Cancer Control (UICC). Ethical approval for human subjects was obtained from the research ethics committee of Zhongshan Hospital of Fudan University. The clinical characteristics of all the patients are listed in Table 1.

Most patients were treated with systemic chemotherapy or traditional Chinese medicine according to their clinical conditions. After treatment conclusion, follow-up was conducted every 6 months, in which patients were monitored by chest, abdomen, and pelvic enhanced CT scanning. Endoscopy was performed annually. Patients with confirmed cancer recurrence received further treatment. OS was defined as the time from the beginning of the study to patient death or study termination, while DFS was defined as the time from the beginning of the study to tumor recurrence or study termination. The 3- and 5-year disease-free survival rates were 8.33% and 6.94%, while the 3- and 5-year overall survival rates were 36.11% and 27.78%.

Cancer tissue and adjacent normal tissue were formalin-fixed, paraffin-embedded, and made into tissue microarrays (TMAs) after hematoxylin and eosin (HE) staining and histopathology-guided locations. Five-micron-thick sections of TMA were deparaffinized and rehydrated, then subjected to high-temperature antigen retrieval via microwave in 0.1 M citrate solution (pH 6.0) for 15 min. The sections were incubated with mouse anti-IRF-2 antibody (Abcam, Cambridge, UK) and anti-FAM123B (Abcam, Cambridge, UK) overnight at 4 °C. The TMA sections were then incubated for 30 min with secondary antibody at room temperature and immunostained by the avidin–biotin complex technique using 3,3'-diaminobenzidine. Hematoxylin was used as a counterstain.

Two pathologists evaluated immunohistochemical staining. The interpretation of immunoreactivity was calculated by analyzing the extent and intensity of staining positivity of cells: “ ≤ 5% cell positivity” or “negative staining” = 0; “6–20% cell positivity” or “light staining” = 1; “21–50% cell positivity” or “mild staining” = 2; “ > 50% cell positivity” or “intense staining” = 3. The total score was the product of the two. The final score was determined by subtracting the score of the cancer tissue from the score of the adjacent tissue. Values greater than 2.5 were considered low expression in IRF-2, while values greater than 2 were considered low expression in AMER-1. All other values were considered high expression.

Human GC cell lines MKN-45 and SGC-7901 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Science, Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China, and cultured in RPMI 1640 medium (HuClone, USA) supplemented with 10% fetal bovine serum (FBS, Corning, USA) at 37 °C in an incubator containing 5% CO₂.

For the experiments utilizing overexpression, the IRF-2 full-length sequence was synthesized and subcloned into the expression vector CMV-MCS-3XFlag-PGK-Puro (Genechem, China). MKN45 cells were transfected with CMV-IRF2-3XFlag-PGK-Puro according to the manufacturer’s instructions. For the knockdown experiments, short hairpin RNA for IRF-2 (shIRF-2) was generated by Genechem (China) and inserted into the pHY-LV-KD1.4 lentiviral shRNA vector (Hanyinbt, China). SGC-7901 cells were transfected with lentiviral shIRF-2 and subjected to selection with puromycin to establish a stable cell line. Stable monoclonal cell lines with up-regulated and down-regulated IRF-2 were screened. The efficacy of overexpression and knockdown of IRF-2 were verified using real-time PCR and western blotting.

For overexpression experiments, the AMER-1 full-length sequence was synthesized and subcloned into a pcDNA3.1 vector (Genechem, China). MKN45 cells were transfected with pcDNA3.1-IRF-2 using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. For the knockdown experiments, SGC-7901 cells were transfected with AMER-1 siRNA according to the manufacturer’s instructions (Genechem, China).

Protein extraction and western blot analysis were performed according to the standard protocols using antibodies against IRF-2 (Abcam, USA), AMER-1 (Abcam, USA), CD44 (EPITMICS, USA), c-myc (Abcam, USA), β-catenin (CST, USA), OCT-4 (Abcam, USA), and SOX-2 (Abcam, USA). β-actin (Abcam, USA) was used as the loading control.

Total RNA was extracted from the cells and tissues using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. A total of 0.5 μg RNA from each sample was subjected to reverse transcription to obtain cDNA using a SuperScript™ III First-Strand Synthesis System Kit (Thermo Fisher Scientific). The resulting cDNA was diluted 100-fold and applied to a real-time PCR (RT-PCR) assay using a Real-time PCR System (Applied Biosystems, USA) with a SYBR Green PCR Master Mix kit (TaKaRa, Japan) following the manufacturer’s protocol. The 2^−ΔΔCt method was used to analyze the relative fold changes. The experiments were performed in triplicate for each data point.

The AMER-1 primers used for PCR were 5'-GGGCTGGACCCCACTGT-3′ (forward) and 5’-CTGCTCAACAGCATCTATCG-3 (reverse), while the IRF-2 primers used for PCR were 5-CGAATGCTGCCCCTATCAGA-3’ (forward) and 5’-TCCTACAACTATGATGTTCACCGT-3’ (reverse). GAPDH was used as an internal control and was detected using the following primers: 5’-AATCCCATCACCATCTTCC-3 (forward) and 5-AGTCCTTCCACGACCAA-3 (reverse).

Plate colony formation assay and 5-Ethynyl-2′-deoxyuridine (EdU) assays were conducted according to standard protocols. Briefly, 500 cells/well were seeded in 6-well plates for the plate colony formation assay. The cells were mixed and cultured for 2 weeks in a culture medium with 10% FBS. Clusters containing more than 30 cells were counted as single colonies. A Cell-Light™ EdU Apollo®488 In Vitro Imaging Kit (RiboBio, China) was used to measure cell proliferation. Images of the cells were obtained using a Nikon microscope (Nikon, Japan). All experiments were repeated three times.

Cells were seeded in a 96-well plate at a concentration of 1500 cells per well. All assays were performed in triplicate. At 1, 2, 3, 4, and 5 days after transfection, the cell proliferation assay was performed by adding 10 μl cell counting kit 8 (CCK8) solution (Beyotime, Shanghai, China) to each well. After incubation at 37 °C for 2 h, cell absorbance was measured at a 450 nm wavelength using a microplate reader (Flexstation III ROM V2.1.28, USA).

Twenty million OE-IRF2-MKN45 cells were grown and washed, then crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched by adding glycine to a final concentration of 0.15 M for 5 min at room temperature. Crosslinked cells were washed with ice-cold PBS, the supernatant was discarded, and the pellets were flash-frozen in liquid nitrogen and stored at − 80 °C.

For each sample, 20 million fixed cells were lysed to prepare nuclear extracts. After chromatin shearing by sonication, lysates were incubated overnight at 4 °C with protein A Dynabeads coupled with 5 μg of antibody. After immunoprecipitation, the beads were recovered using a magnet and then washed. DNA was eluted and cross-links reverted at 65 °C for 4 h, then purified using the QIAGEN Kit. DNA was quantitated using the Qubit® dsDNA HS assay and a Qubit® 2.0 Fluorimeter (Invitrogen). For ChIP-Seq, 5 ng of purified ChIP DNA was used to generate the sequencing library using an NEB kit and sequenced with the Illumina HiSeq X Ten. Each sample was tested at least three times.

For ChIP-seq data analysis, FastQC software was used to evaluate the quality of the original data. The original data were then compared to the reference genome using BWA or Bowtie2 software. MACS was used for peak calling, genome location annotation of peak mining, motif analysis of peak area, and GO and KEGG enrichment analysis of the target genes.

Then, AMER-1-wild and -mut were inserted into the pGL3 promoter vector (GenScript Co., Ltd., Nanjing, China), which was transfected into 7901 and MKN-45 cells using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.), along with IRF-2 overexpression vectors or NC vectors. The cells were seeded in 24-well plates. After 48 h, firefly luciferase signals and Renilla luciferase (internal reference) were detected using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA.) according to the manufacturer’s protocol. All experiments were performed in triplicate.

Ten female BALB/c nude mice (4 to 6 weeks old and weighing 18 to 20 g) were obtained from the Shanghai Experimental Animal Center (Shanghai, China). shIRF2-SGC7901 and NC-SGC7901 cells (2 × 10⁶) were harvested and injected subcutaneously into nude mice (five mice per group). Tumor growth was quantified every 2 days after tumor formation, and tumor volumes were calculated as length × width² × 0.5. After about 23 days, tumors were removed when the mice were sacrificed. Tumor tissues were then formalin-fixed, paraffin-embedded, HE stained, and immunohistochemically stained to measure the expression levels of IRF-2, AMER-1, and CD44. All xenograft experiments were approved by the Animal Experiments Ethics Committee of Zhongshan Hospital of Fudan University.

The co-expression relationship between IRF-2 and AMER-1 at the mRNA level was investigated using the GEPIA database (http://​gepia.​cancer-pku.​cn/​index.​html) [17]. The Spearman correlation coefficient R was calculated to describe the relationship. Samples were retrieved from the stomach cancer dataset (STAD) of The Cancer Genome Atlas (TCGA; http://​tcga-data.​nci.​nih.​gov/​tcga/​) database. There were 370 cases comparing the IRF-2 and AMER-1 expression levels in terms of log transformed transcripts per million (TPM) values.

The expression level of IRF-2 was examined in TMAs containing 72 pairs of GC tissues and normal adjacent tissues by IHC analysis. Immunohistochemical analyses revealed that IRF-2 was mostly located in the cytoplasm and downregulated in human GC tissues compared with normal adjacent tissues (P < 0.001; Fig. 1A, B). The average score of IRF-2 was 3.90 ± 1.56 in GC tissues and 6.35 ± 1.65 in normal adjacent tissues.

To determine the relationship between the expression level of IRF-2 and the clinical characteristics of GC patients, we collected the patients’ data and summarized them in Table 1. There was no correlation between IRF-2 expression and clinical characteristics including age, sex, tumor size, invasive depth, lymph node metastasis, tumor position, and TNM stage (P > 0.05).

A Kaplan–Meier analysis and log-rank test were used to evaluate the influence of IRF-2 on survival. We found that the IRF-2 expression level was significantly positively correlated with patients’ overall survival time (OS) (P < 0.001, Fig. 1C) and cancer-free survival time (DFS) (P = 0.014, Fig. 1D), indicating that higher IRF-2 expression correlated with longer DFS and OS. Tumor size, TNM stage, invasive depth, and lymph node metastasis were unfavorable predictors for OS. Tumor size, TNM stage, invasive depth, lymph node metastasis, and distant metastases were unfavorable predictors for DFS. IRF-2 was a favorable predictor for both OS and DFS of GC (Table 2). Considering that the invasive depth, lymph node metastasis, and distant metastases were included in the TNM stage, we only included tumor size, TNM stage, and expression of IRF-2 in the multivariate analysis. This analysis found that IRF-2 was an independent prognostic factor for OS (P < 0.001) and DFS (P = 0.002).

The stable cell lines with overexpression of IRF-2 in MKN-45 and knockdown of IRF-2 in SGC-7901 have been constructed and validated in our previous studies [16]. Colony formation assays showed that colony formation ability decreased following IRF-2 overexpression in MKN-45 cells, whereas colony formation ability increased following IRF-2 knockdown in SGC-7901 cells (Fig. 2A). Further, IRF-2 overexpression remarkably decreased the proliferation of MKN-45 cells, whereas knockdown of IRF-2 increased cell proliferation (Fig. 2B). Similarly, EdU assays also showed that IRF-2 overexpression inhibited GC cell proliferation, whereas its knockdown promoted GC cell proliferation (Fig. 2C).

We further explored whether IRF-2 affects GC growth in vivo. SGC-7901 cells stably transfected with sh-IRF-2 and empty vectors were injected into nude mice. Twenty-three days after the injection, tumors from the sh-IRF-2 group were significantly larger than those from the control group (Fig. 2D). These findings indicate that IRF-2 can negatively affect GC cell proliferation both in vitro and in vivo.

We applied ChIP-Seq to investigate the potential target and binding sites of IRF-2 in GC and found 18,565 peaks (Fig. 3A). GO and KEGG enrichment analysis of target genes were used to explore the signaling pathways may be affected by IRF-2. The ten pathways in which IRF-2 was most affected by GO analysis included the tumor necrosis factor-mediated signaling pathway and the Wnt/β-catenin signaling pathway, both of which were related to tumor development and progression (Fig. 3B). KEGG analysis also showed that IRF-2 may affect several cancer pathways (Fig. 3C). Combined with the previously reported results of microarray assays [16], we found that IRF-2 can inhibit the Wnt/β-catenin signaling pathway by directly targeting the AMER-1 transcription start domain. We also found that IRF-2 may act on the promoter region of AMER-1 to promote transcription by Chip-Seq (Fig. 3D). Possible binding sites of IRF-2 were identified using the JASPAR 2020 database (Fig. 3E) [18] and there were two predicted binding sites in the AMER-1 transcription start domain (Fig. 3F), which is consistent with our ChIP-Seq results. To determine whether IRF-2 binds to the AMER-1 promoter, we performed luciferase assays. The results showed that IRF-2 significantly upregulated the luciferase activity of AMER-1-promoter-WT, but not AMER-1-promoter-Mut1 nor AMER-1-promoter-Mut2 (Fig. 3G). This suggests that IRF-2 binds to the AMER-1 promoter in GC.

We verified the AMER-1 expression and the key factors affecting the Wnt/β-catenin signaling pathway, including CD44, c-myc, and β-catenin, in lentivirus-infected cell lines. AMER-1 expression increased after IRF-2 was overexpressed, while AMER-1 expression decreased when IRF-2 was downregulated, at both the protein and mRNA levels (Fig. 4A, B). IRF-2 expression was also negatively correlated with the indices of stem cell-like features, including OCT-4, SOX-2, CD44, and c-myc (Fig. 4A). To further evaluate the relationship between IRF-2 and AMER-1 in GAC patients and xenografted tumor tissues in nude mice, we examined the expression levels of AMER-1 by immunohistochemical assay, using anti-AMER-1 antibody in the same TMA specimens and xenografted tumor tissues. The immunohistochemical scores showed positive correlations between AMER-1 and IRF-2 scores, both in TMA specimens (r = 0.58, P < 0.001; Fig. 4C) and xenografted tumor tissues (r = 0.59, P < 0.001; Fig. 4D). A significant inverse correlation was also found between AMER-1 expression and CD44 (r = − 1.55, P = 0.009; Fig. 4D). A positive correlation was also found between the expressions of IRF-2 and AMER-1 on the GEPIA (r = 0.19, P < 0.001; Fig. 4E).

To determine whether IRF-2 regulated the expression of the Wnt/β-catenin signaling pathway by targeting AMER-1, we knocked down the expression of AMER-1 in MKN-45 cells with or without overexpressed IRF-2. We found that key molecules in the Wnt/β-catenin signaling pathway and stem cell-like features were upregulated when AMER-1 was downregulated, regardless of the expression of IRF-2 (Fig. 5A). Similar results were also observed in the cytofunctional experiments. Knocking down the expression of AMER-1 led to increased colony formation ability (Fig. 5B) and promoted GC cell proliferation (Fig. 5C), despite overexpression of IRF-2. Similarly, inhibition of the Wnt/β-catenin signaling pathway was observed, in both western blot analyses and cytofunctional experiments, when AMER-1 was upregulated, regardless of IRF-2 expression (Fig. 5D–F). All the results indicated that the ability of IRF-2 to inhibit the Wnt/β-catenin signaling pathway was dependent on AMER-1 regulation.