The Mechanism Underlying MFGE8-mediated Promotion Of Peritoneal Metastasis And Its Role As A Therapeutic Target For Gastric Cancer

Objective: Gastric cancer is a highly prevalent cancer worldwide.Although the combination of surgery,radiotherapy,chemotherapy,and immunotherapy has improved patient survival,most patients continue to experience recurrence and metastasis,leading to poor prognosis.Peritoneal metastasis is one of the most common forms of gastric cancer metastasis,occurring in up to 14% of the newly diagnosed gastric cancer patients.It is also the most common site of recurrence after radical resection in gastric cancer patients.Because of the lack of effective treatment,the median survival of patients with peritoneal metastases is 3–6 months.Therefore,it is imperative to identify reliable therapeutic targets for peritoneal metastasis of gastric cancer.Milk fat globule epidermal growth factor 8(MFGE8)is a secreted glycoprotein that is widely expressed in vivo.Studies have shown that MFGE8 is involved in the occurrence,development,and metastasis of various cancers,including lung squamous cell carcinoma,glioma,oral squamous cell carcinoma,prostate cancer,bladder cancer,pancreatic ductal adenocarcinoma,hepatocellular carcinoma,triple-negative breast cancer,colorectal cancer,papillary thyroid cancer,ovarian epithelial cancer,angiosarcoma,esophageal cancer,urothelial bladder cancer,cholangiocarcinoma,and malignant melanoma.Further studies on the role and mechanism of MFGE8 in gastric cancer and its importance as a potential biomarker for peritoneal metastasis in gastric cancer need to be explored.Studies have shown that 3,3’-diindolylmethane(DIM),a natural phytochemical derived from cruciferous vegetables,leads to the death of tumor cells but only has a weak cytotoxic effect on normal cells.DIM is very safe,thus making it a potential candidate as an anti-tumor drug.However,the role of DIM in peritoneal metastasis of gastric cancer and its relationship with MFGE8 remain unclear.Methods: 1.Potential biomarkers for peritoneal metastasis of gastric cancer were identified by screening the Gene Expression Omnibus(GEO)and Gene Expression Profiling Interactive Analysis 2(GEPIA 2)databases.2.MFGE8 expression and its association with clinical prognosis were analyzed using the gastric cancer dataset in The cancer genome atlas(TCGA)database.3.The association of MFGE8 with clinicopathologic parameters and survival rates were analyzed using the gastric cancer datasets in the GEO and TCGA databases.4.The association of MFGE8 expression with clinicopathologic parameters and survival rates were assessed using immunohistochemistry in surgically excised tissue samples of 90 patients with gastric cancer.5.Morphologic differences between MKN-45 cells,a human gastric cancer cell line,and MKN-45 P cells,an MKN-45 clone with high peritoneal metastatic potential,were evaluated using light microscopy.Colony formation,adhesion,and transwell assays were used to detect functional differences between the two cell lines.6.MFGE8 expression in human gastric epithelial cell lines GES-1,MKN-45,and MKN-45 P was determined using western blotting.7.Compounds that inhibited MFGE8 expression in gastric cancer were identified using the CMap database.8.Molecular docking analysis was conducted to examine MFGE8-DIM interaction.9.The gastric cancer cells were transfected with si RNA or treated with recombinant human MFGE8 protein(rh MFGE8)and DIM.The transfection efficiency of si RNA was verified by Real Time PCR and western blotting.10.The proliferation ability of MKN-45 and MKN-45 P cells was determined with the Cell Counting Kit-8(CCK-8)and colony-formation assays.11.The ability of MKN-45 and MKN-45 P cells to adhere to human peritoneal mesothelial HMr SV5 cells was determined by the adhesion assay.12.The migration and invasion abilities of MKN-45 and MKN-45 P cells were determined by the transwell assay.13.The Kyoto encyclopedia of genes and genomes(KEGG)and Gene Set Enrichment Analysis(GSEA)were used to analyze pathways enriched in cells expressing higher MGFE8.14.The gastric cancer dataset in the TCGA database was used to examine the correlation between MFGE8 expression and molecules related to epithelial–mesenchymal transition(EMT).15.The molecules related to the mitogen-activated protein kinases/extracellular signal-regulated kinase(MAPK/ERK)signaling pathway and EMT in MKN-45 and MKN-45 P cells were detected by western blotting.16.The peritoneal metastasis model of gastric cancer in nude mice was established using MKN-45 P cells,and the number of gastric cancer peritoneal metastases was compared among mice in the following treatment groups: DIM,rh MFGE8,DIM plus rh MFGE8,and control.17.Expression levels of MFGE8,E-cadherin,N-cadherin,vimentin,metalloproteinase protein 9(MMP9)in the peritoneal metastatic tumor tissues of nude mice were determined using immunohistochemistry.Results:Part 1: Research on the expression and clinical significance of MFGE8 in gastric cancer based on bioinformatics analysis and immunohistochemistry.1.1 Based on the screening results of gastric cancer datasets in the GEO and GEPIA 2databases,MFGE8 is one of the potential biomarkers for peritoneal metastasis of gastric cancer.1.2 MFGE8 expression was markedly increased in gastric cancer.The overall survival,disease-specific survival,and progression-free interval of patients with high MFGE8 expression were considerably lower than those of patients with low MFGE8 expression.In the TCGA gastric cancer dataset,MFGE8 expression was greatly associated with tumor histological grade.In the GSE62254 dataset,MFGE8 expression was significantly associated with pathological T stage,pathological N stage,pathological M stage,tumor stage,liver metastasis,peritoneal metastasis,ascites,and other site metastasis.Univariate and multivariate Cox regression analysis revealed that the high expression of MFGE8 in the TCGA gastric cancer dataset and the GSE62254 dataset was an independent risk factor for the prognosis of gastric cancer patients.1.3Immunohistochemical analysis of pathological sections from 90 gastric cancer patients demonstrated that MFGE8 expression was significantly associated with pathological T stage,pathological N stage,and tumor stage.The overall survival of patients with high MFGE8 expression was substantially shorter than that of patients with low MFGE8 expression.Part 2: Role and mechanism of MFGE8 in peritoneal metastasis of gastric cancer2.1 Microscopic examination of the human gastric cancer cell line MKN-45 showed mostly spherical semi-suspended cells growing in clusters,whereas the high-potential peritoneal dissemination human gastric cancer cell line MKN-45 P showed a large number of spindle-shaped adherent cells.2.2 Colony formation,adhesion,and transwell assays showed that MKN-45 P cells had stronger proliferation,adhesion,migration,and invasion abilities than MKN-45 cells.2.3 Western blot analysis showed that the expression of MFGE8 in gastric cancer cell lines was higher than in human gastric mucosal epithelial cell line GES-1.Moreover,the expression of MFGE8 in MKN-45 P cells was higher than in MKN-45 cells.2.4 Treatment with rh MFGE8 increased the proliferation,adhesion,migration,and invasion abilities of MKN-45 cells.2.5Knockdown of MFGE8 expression by si RNA decreased the proliferation,adhesion,migration,and invasion abilities of MKN-45 P cells.2.6 KEGG and GSEA analysis showed that MFGE8 might be enriched in MAPK signaling pathway.2.7 TCGA gastric cancer dataset analysis showed that expression of MFGE8 was negatively correlated with the expression of E-cadherin and positively correlated with the expression of N-cadherin,vimentin,and MMP9.2.8 Western blot analysis showed that the phosphorylation level of ERK in MKN-45 cells was increased after treatment with rh MFGE8,while the amount of total ERK protein was unchanged.Furthermore,expression of E-cadherin decreased,whereas the expression of N-cadherin,vimentin,and MMP9 increased.The expression of MFGE8 in MKN-45 P cells was decreased after transfection with si RNA of MFGE8 and accompanied by a decrease in the phosphorylation level of ERK,although the total ERK protein level remained unchanged.Simultaneously,the expression of E-cadherin increased and the expression of N-cadherin,vimentin,and MMP9 decreased.Part 3: Effect of DIM on peritoneal metastasis of gastric cancer through inhibition of MFGE8 expression3.1 Screening of the CMap database suggested that DIM could be an inhibitor of MFGE8 expression in gastric cancer.3.2 Molecular docking showed that MFGE8 could potentially bind well to DIM.3.3 Western blot analysis showed that DIM could inhibit the expression of MFGE8 protein in MKN-45 and MKN-45 P cells.3.4 CCK-8 assays showed that DIM inhibited the proliferation of MKN-45 and MKN-45 P cells in a doseand time-dependent manner.3.5 The results of colony formation,adhesion,and transwell assays showed that the proliferation,adhesion,migration,and invasion abilities of MKN-45 and MKN-45 P cells were substantially decreased after DIM treatment.Combined treatment with rh MFGE8 and DIM ablated the increase in proliferation,adhesion,migration,and invasion abilities induced by rh MFGE8 alone.3.6 After DIM treatment,activation of the MAPK/ERK signaling pathway was inhibited in MKN-45 and MKN-45 P cell lines.Expression of E-cadherin increased,and expression of N-cadherin,vimentin,and MMP9 decreased.Combined treatment with rh MFGE8 and DIM ablated the activation of the MAPK/ERK pathway and EMT induced by rh MFGE8 alone.3.7 The number of peritoneal metastases of gastric cancer in nude mice in the DIM injection group was lower than that in the control group.In contrast,the number of peritoneal metastases of gastric cancer in the rh MFGE8 injection group was higher than that in the control group.The number of peritoneal metastases of gastric cancer in nude mice injected with rh MFGE8 and DIM together was lower than that in mice injected with rh MFGE8 alone.3.8 Immunohistochemistry results showed that expression of MFGE8 decreased,expression of E-cadherin increased,and expression of N-cadherin,vimentin,and MMP9 decreased in the DIM injection group.Conversely,expression of E-cadherin decreased and expression of N-cadherin,vimentin,and MMP9 increased in the rh MFGE8 injection group.Co-injection of rh MFGE8 and DIM reversed EMT induced by rh MFGE8 alone.Conclusion: Through bioinformatic analysis and immunohistochemical verification,we confirmed that MFGE8 was upregulated in gastric cancer,and that its high expression was positively correlated with poor prognosis and peritoneal metastasis in gastric cancer patients.The high expression of MFGE8 may be an independent predictor of poor prognosis.MFGE8 enhances the proliferation,adhesion,migration,and invasion of gastric cancer cells by activating the MAPK/ERK signaling pathway,thereby promoting their EMT process.DIM can inhibit expression of MFGE8,reduce activation of the MAPK/ERK signaling pathway,limit the proliferation,adhesion,migration,and invasion of gastric cancer cells,and reverse EMT—thereby delaying peritoneal metastasis of gastric cancer.These results suggest that MFGE8 may be a biomarker for peritoneal metastasis of gastric cancer and that DIM may treat peritoneal metastasis of gastric cancer by targeting MFGE8.This study provides more evidence for target prediction and clinical treatment of peritoneal metastasis of gastric cancer.