TNF−α Secreted from Macrophages Increases the Expression of Prometastatic Integrin αV in Gastric Cancer

The tumor microenvironment comprising blood vessels, fibroblasts, immune cells, and the extracellular matrix surrounding cancer cells, has recently been targeted for research in cancer therapy. We aimed to investigate the effect of macrophages on the invasive ability of gastric cancer cells, and studied their potential mechanism. In transcriptome analysis, integrin αV was identified as a gene increased in AGS cells cocultured with RAW264.7 cells. AGS cells cocultured with RAW264.7 cells displayed increased adhesion to the extracellular matrix and greater invasiveness compared with AGS cells cultured alone. This increased invasion of AGS cells cocultured with RAW264.7 cells was inhibited by integrin αV knockdown. In addition, the increase in integrin αV expression induced by tumor necrosis factor-α (TNF-α) or by coculture with RAW264.7 cells was inhibited by TNF receptor 1 (TNFR1) knockdown. The increase in integrin αV expression induced by TNF-α was inhibited by both Mitogen-activated protein kinase (MEK) inhibitor and VGLL1 S84 peptide treatment. Finally, transcription of integrin αV was shown to be regulated through the binding of VGLL1 and TEAD4 to the promoter of integrin αV. In conclusion, our study demonstrated that TNFR1–ERK–VGLL1 signaling activated by TNF-α secreted from RAW264.7 cells increased integrin αV expression, thereby increasing the adhesion and invasive ability of gastric cancer cells.


Introduction
Gastric cancer is known to be highly heterogeneous in both the intratumoral and intertumoral environments. One aspect of this heterogeneity occurs within the tumor microenvironment, which is highly permeated by cancer-related fibroblasts, immune cells, and stromal components [1]. One characteristic of the tumor microenvironment in gastric cancer is chronic inflammation derived from infection, such as Helicobacter pylori, which promotes cell survival and causes the upregulation of pathways that activate stem cell and epithelial cell proliferation [2]. Helicobacter pylori and other pathogens impair M1 macrophage responses, thereby leading to an M2-like state; these pathogens increase the rate of reactive oxygen species-induced macrophage apoptosis, which exacerbates the risk of disease progression [2]. Infection by Helicobacter pylori induces inflammation in the stomach microenvironment and is associated with the induction of cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β, and interleukin-6, which lead to gastric cancer [3].
Macrophages are a major component of the tumor microenvironment and regulate various aspects of immunity. Depending on their activation status, macrophages can exert dual effects on tumorigenesis, either antagonizing the cytotoxic activity of immune cells or strengthening antitumor reaction [4]. Macrophages promote invasion and metastasis from the primary tumor sites through their ability to let cancer cells engage in an autocrine loop that promotes cancer cell migration [5]. Macrophage-derived cathepsins, SPARC, or CCL18 improve tumor cell adhesion to extracellular matrix proteins and promote tumor cell migration [5]. It has been reported that TNF-α, an inflammatory cytokine secreted from macrophages, promotes cancer cell proliferation, migration, and invasion in gastric cancer, colon cancer, breast cancer, and kidney cancer [3,[6][7][8][9]. TNF is involved in various intracellular signaling mechanisms and has been reported to regulate the expression of the integrin family proteins in various diseases. TNF-α increased αVβ3 expression in human chondrosarcoma cells [10]. Fibroblast paracrine TNF-α increased integrin β5 expression in idiopathic pulmonary fibrosis [11]. TNF-α induced the activation of β-integrin function [12].
Integrins are a group of transmembrane proteins that act as cell-matrix adhesion receptors for transmitting signals and regulating various biological processes. To date, 18 α and 8 β subunits have been identified; these can directly form 24 known heterodimers and each α/β combination confers binding specificity of the extracellular domains to different ligands [13]. Many studies have also demonstrated the possibility of integrins as therapeutic targets in cancer treatment [14]. Integrins have been reported as diagnostic and prognostic markers for gastric cancer through various analyses of integrin expression and prognosis [15,16]. Integrin αV is a member of the integrin alpha chain family and can associate with up to five beta subunits to form αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 integrin receptors [17]. Integrin αV promotes the growth, migration, and invasion of gastric cancer cells [17]. Even through integrin αV is known as a target gene for FOSL1, TAZ, and SIN3B, there have been no reports of the mechanism of the regulation of integrin αV expression in gastric cancer [18][19][20].
In this study, we analyzed the pathways associated with the changes in gene expression induced by the coculture of macrophages cells and gastric cancer cells and identified integrin αV. In addition, the mechanism via which TNF-α-induced integrin αV promotes the metastasis of gastric cancer cells was verified.

Integrin αV was Identified as a Gene with Increased Expression in AGS Cells Cocultured with RAW264.7 Cells
First, we performed RNA-Seq to confirm changes in the expression of genes in the relevant pathway in human gastric cancer cells (AGS cells) and AGS cells cocultured with macrophages (RAW264.7 cells) ( Figure 1A). In the gene ontology enrichment analysis of the 160 genes that were increased in AGS cells cocultured with RAW264.7 cells, pathways related to immune response, cell adhesion, and cytokine response were enriched ( Figure 1B). STRING analysis revealed the strong binding of TNF-α signaling-related genes ( Figure 1C). We confirmed that the expression of TNFSF10, TNFRSF9, and integrin αV were top-ranked genes involved in TNF-α signaling ( Figure 1D); among them, integrin αV, which was reported to play an important role in cell adhesion, was selected for further study.

Integrin αV Regulated the Adhesion and Invasion of AGS Cells Cocultured with RAW264.7 Cells
Next, to verify the effect of macrophages on the adhesion and invasion of gastric cancer cells, the adhesion of AGS cells to the extracellular matrix (ECM) proteins, such as vitronectin, collagen, and fibronectin, was examined. AGS cells cocultured with RAW264.7 cells increased cell adhesion to ECM compared to the cells cultured with AGS alone (Figure 2A). The invasion of AGS and NUGC3 cells was also increased by coculture with RAW264.7 cells ( Figure 2B,C). Furthermore, the protein expression of integrin αV was increased in AGS and NUGC3 cells by being cocultured with RAW264.7 cells ( Figure 2D). Next, to determine the effect of integrin αV on the metastasis of gastric cancer cells, integrin αV was transfected into AGS cells ( Figure 2E). Overexpression of integrin αV increased the adhesion and invasion of AGS cells ( Figure 2F,G). By contrast, the invasion of AGS cells increased via coculture with RAW264.7 cells was also inhibited in AGS cells treated with siRNA of integrin αV or siIntegrin αV ( Figure 2H). These results suggest that integrin αV is involved in the macrophage-induced invasion of gastric cancer cells.  The bars indicate the S.D.; the asterisks denote significant differences (*** p < 0.001, ** p < 0.01, * p < 0.05) between the means of two groups.

Integrin αV Expression Was Increased in Gastric Cancer Cells through TNF-α/TNFR1/ERK Signaling
The effect of TNF-α, a cytokine secreted from macrophages, on integrin αV expression was investigated. TNF-α increased integrin αV expression in AGS, NCI-N87, and NUGC3 human gastric cancer cells ( Figure 3A). Subsequently, RT-PCR was performed to examine the expression of TNF receptors in gastric cancer cell lines. The expression of TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) was increased in AGS, NCI-N87, NUGC3, and MKN1 cells; meanwhile, the receptor TNFRSF9 was expressed only in MKN1 cells ( Figure 3B). This increase in integrin αV expression induced by TNF-α was inhibited by treatment with siTNFR1 but not siTNFR2 ( Figure 3C). Furthermore, TNFR1 knockdown inhibited the increased integrin αV expression induced by coculturing with RAW264.7 cells ( Figure 3D). TNF-α induced phosphorylation of ERK in AGS cells ( Figure 3E), however this was inhibited by TNFR1 knockdown ( Figure 3F). Pretreatment with PD98059, a MEK inhibitor, reduced this increase in integrin αV expression induced by TNF-α ( Figure 3G). Furthermore, the knockdown of RSK2, a downstream target of ERK, inhibited the increase in integrin αV expression induced by TNF-α ( Figure 3H). These results suggest that TNF-α activates TNFR1 and then phosphorylates ERK, thereby regulating integrin αV expression.

VGLL1 Regulates the Invasion of Gastric Cancer Cells through Integrin αV
We next investigated the effect of VGLL1, which is known as a downstream protein of RSK2, on the increase in integrin αV expression by TNF-α. The TNF-α-induced expression of integrin αV was shown to be suppressed in AGS cells treated with siVGLL1 compared with cells treated with siScramble ( Figure 4A). In addition, the expression of integrin αV was shown to be suppressed even in the presence of TNF-α by the treatment with VGLL1 S84 peptide, which inhibits VGLL1 phosphorylation ( Figure 4B). In addition, the expression of integrin αV increased by coculturing of AGS cells and RAW264.7 cells was inhibited by VGLL1 S84 peptide ( Figure 4C). Finally, we observed that the VGLL1 S84 peptide inhibited the invasion of gastric cancer cells increased by coculture with RAW264.7 cells ( Figure 4D).
These results suggest that TNF-α in macrophages induces the phosphorylation of VGLL1, which regulates the invasion of gastric cancer cells. Cell invasion was analyzed using the SRB assay (n = 3). ** p < 0.01, *** p < 0.001.

Transcription of Integrin αV Is Regulated by VGLL1 and TEAD4
VGLL1 is known to bind to the transcription factor TEAD4 to regulate the expression of target genes. Therefore, it was examined whether TEAD4 regulates the transcription of integrin αV. The increased mRNA expression and promoter activity of integrin αV by TNF-α were shown to be inhibited by siTEAD4 ( Figure 5A,B). For the binding of TEAD4 in the promoter region of integin αV, two TEAD4 binding sites (−577/−567, −513/−503) were previously identified [19] and Chromatin Immunoprecipitation (Chip) assays indicate that TEAD4 and VGLL1 bind to the first TEAD4 binding site of the promoter of integrin αV ( Figure 5C). Furthermore, when an integrin αV promoter with mutated or deleted TEAD4 binding site was constructed ( Figure 5D) and its binding activity was tested, the increase in integrin αV promoter activity by VGLL1-TEAD4 binding was suppressed when the first TEAD4 binding site was mutated or deleted but remained unaffected when the second TEAD4 binding site was mutated ( Figure 5E). These results suggest that the TNF-α-induced integrin αV expression is regulated through VGLL1-TEAD4 binding. ; the asterisks denote significant differences (*** p < 0.001, ** p < 0.01, * p < 0.05) between the means of two groups.

Discussion
In this study, we have verified that TNF-α secreted from macrophages promotes cancer metastasis by increasing integrin αV expression in gastric cancer cells. When AGS cells were cocultured with RAW264.7 cells, the cell adhesion and the invasion of AGS cells increased; moreover, the expression of TNF-α-related genes was also increased. In AGS cells, the expression of integrin αV was increased by treatment with TNF-α or co-culturing with RAW264.7 cells. In addition, TNFR1 knockdown in AGS cells also inhibited TNFα-induced integrin αV expression. This supports the existing view that integrin αV is important for the proliferation and metastasis of gastric cancer.
The importance of integrin as a target for cancer treatment has been well recognized for several years, and numerous integrin inhibitors have been developed [21,22]. Various integrin inhibitors are being tested in clinical trials, but successful results have not been obtained. As an intracellular signal transduction mechanism, ECM-ligated integrins initiate prosurvival signaling by BCL-2, FLIP factor-kB, NF-kB, and PI3K-AKT. In an unligated state, the integrins initiate a process known as integrin-mediated death via the cleavage of caspase-8 [21]. Although the regulatory mechanism of integrin in cancer cells has been reported, studies on the mechanism through which integrin expression is regulated by the tumor microenvironment remain uncommon. We demonstrated the regulation of integrin αV expression through the TNF-α-ERK-VGLL1-TEAD4 pathway in the coculture of gastric cancer cells with macrophages cells.
In liver cancer cells, integrin αV dose not bind to YAP, but instead binds to the promoter together with TEAD4 as a target gene for TAZ [19]. In gastric cancer, we confirmed that VGLL1, which has a similar structure to TAZ, binds with TEAD4 to the integrin αV promoter in the presence of TNF-α. The binding site of VGLL1 was identified by mutating or deleting the TEAD4 binding sites in the integrin αV promoter. Furthermore, it was confirmed that the correlation between VGLL1 and integrin αV expression in gastric cancer patients was significant (Supplementary Figure S1). This suggests that the expression of integrin αV can be regulated by PI3K [23], TGF-β [24], TNF-α, which are upstream signaling proteins of VGLL1, in addition to the YAP/TAZ signaling.
The function of TNF in immune cells is generally well established, and studies related to cancer malignancy have also been reported. The mRNA and protein expression of TNF have been detected in malignant and stromal cells from human cancer biopsy samples [25][26][27]; moreover, plasma TNF levels have been found to be increased in some patients with cancer, notably those with poor prognosis [28,29]. TNF in the tumor microenvironment can induce cancer malignancy through genetic damage or directly affect the EMT [25,30]. In human bile duct sarcoma cells, TNF induced DNA and RNA editing enzymes, activation-induced cytidine deaminase, which led to mutations in genes such as TP53 and MYC [31]. TNF antagonists inhibit cytokine and chemokine production, inflammatory cell recruitment, angiogenesis, and extracellular substrate degradation [25,32]. Cancer metastasis suppression experiments using anti-TNF antibodies have been verified by single or combined treatment in various cancer types since they were first reported in 1993 [25]. A Phase 1 study of the anti-TNF antibody infliximab reported disease stabilization in 7 out of 41 patients with progressive cancer [33].
Recently, studies have reported a tumor microenvironment promoting tumor malignancy in several carcinomas. Integrin α11-expressing cancer-associated fibroblast induces breast cancer metastasis via the platelet-derived growth factor receptors (PDGFRs)-JNK pathway [34]. In the analysis of transcriptome data from hypoxic and normoxic cultured human peripheral blood mononuclear cell (PBMC)-derived macrophages, it was found that C-X-C motif chemokine ligand 8 (CXCL8) expression was increased, and CXCL8 is reportedly involved in gastric cancer malignancy [35]. The increased expression of ECM metalloproteinase inducer (EMMPRIN, CD147) induced by the coculture of human renal carcinoma A498 cells and human breast carcinoma MCF-7 cells with human monocytelike U937 cells promotes angiogenesis [36]. When human lung cancer A549 cells and RAW264.7 cells were cocultured, the expression of metastasis-related genes, such as MMPs and VEGF, was increased [37,38].
We confirmed that immune system processes, immune response pathways, and cell adhesion pathways were involved through the coculture of macrophages and gastric cancer cells. However, clinical research and development of therapeutic agents that inhibit substances secreted by macrophages have not yet been explored for patients with gastric cancer. Furthermore, it is necessary to study immunotherapeutic agents that can increase the efficacy of the gastric cancer drugs currently used in clinical practice. We were interested in ascertaining whether the suppression of TNFR in gastric cancer cells could increase the efficacy of anticancer drugs, and it was confirmed that oxaliplatin and cisplatin displayed a greater increase of the inhibitory efficacy of cell proliferation in the siTNFR1-treated group compared with the control group (Supplementary Figure S2). Our study confirmed that the secretion of TNF-α by macrophages promotes the metastasis of gastric cancer cells, thereby providing a basis for the use of TNF/TNFR inhibitors for the development of gastric cancer treatment agents. Consequently, experiments to confirm the effects of the coadministration of anticancer drugs and TNF inhibitors in animal models are required.

Cell Lines
Human gastric cancer cells (AGS, NCI-N87, and NUGC3) were purchased from the Korea Cell Line Bank (Seoul, Korea) and cultured in Roswell Park Memorial Institute -1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. RAW264.7 murine macrophages cells were provided by Dr. Haiyoung Jung (KRIBB, Korea) and cultured in Dulbecco's modified Eagle medium containing 10% FBS and 1% penicillin/streptomycin. All cells were maintained in an incubator at 37 • C with 5% CO 2 .

Transcriptome Analysis
The total RNA extracted from AGS cells and AGS cells cocultured with RAW264.7 cells was constructed into a library using TruSeq Stranded mRNA LT sample prep kit. Sequencing was performed using the Illumina platform (Macrogen, Seoul, Korea). The dataset is available in GEO DataSet of the NCBI Database under the accession number GSE220531.

Western Blotting
Cells were lysed in RIPA buffer (Millipore, Billerica, MA, USA) containing protease inhibitor cocktail (Roche, Basel, Switzerland). The lysates were quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA) and the obtained proteins (20 µg) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The proteins were identified using the following antibodies: anti-VGLL1

Cell Adhesion and Invasion Assay
For the adhesion assay, cells were seeded in 96-well plates coated with BSA (1%), vitronectin (10 µg/mL), fibronectin (10 µg/mL), and collagen (50 µg/mL). The cells were allowed to attach for 30 min and nonadherent cells were removed by washing with 0.1% BSA. For the invasion assays, we used chambers with a 0.8 µm pore membrane in 24-well inserts (BD Biosciences). We seeded the cells into the upper part of each chamber, which was coated with Matrigel (BD Biosciences), and the lower compartments were filled with the medium. To analyze the adherent or invasive cells, 0.4% sulforhodamine B (SRB) was used to stain the cells after fixation with 10% formalin, as described previously [39]. Unbound dye was washed with 0.1% acetic acid; protein-bound dye was dissolved in 10 mM Tris, and its optical density was measured at 540 nm.

Luciferase Assay
The activity of the integrin αV promoter was determined by the dual-luciferase reporter system (Promega, Madison, WI, USA). Cells were transfected with pGL4.17-Integrin αV promoter-luc and pRL-SV40 encoding firefly luciferase (Renilla-Luc) using Turbofect Transfection Reagent (Thermo Fisher). Cells were harvested 48 h after transfection and lysed in 1× passive lysis buffer (Promega). Luciferase activity was measures using a multi-mode microplate reader (SYNERGY/HTX, BioTek, Winooski, VT, USA) and then normalized to the activity of Renilla luciferase.

Conclusions
The coculture of RAW264.7 cells and AGS cells increased the expression of integrin αV through the TNFR-ERK-VGLL1 pathway; this subsequently increased the adhesion and invasion of gastric cancer cells. Moreover, this effect was also observed after treatment with TNF-α; therefore, we suggest that TNF-α secreted by macrophages plays an important role in the metastasis of gastric cancer cells.

Data Availability Statement:
The dataset is available in the NCBI Database of GEO datasets under the data series accession number GSE220531.