title: CD74 and macrophage migration inhibitory factor as therapeutic targets in gastric cancer.
abstract: OBJECTIVE
  To investigate the relationship and molecular features of CD74/macrophage migration inhibitory factor (MIF)/Toll-like receptor 4 (TLR4) in gastric cancer.
  
  
  METHODS
  CD74, MIF and TLR4 expression in the paraffin-embedded sections of gastric cancer from 120 patients were detected by immunohistochemical staining. Knock down of CD74 expression in gastric cancer cell line MKN-45 was performed by lentivirus transduction and detected by Western blotting. MKN-45 cell proliferation assay under the stimulants was measured by the cell counting kit 8 (CCK8) assay and MIF concentration in the culture medium was detected by enzyme-linked immunosorbent assay. Surface staining of CD74 in the MKN-45 cell line under the stimulation of lipopolysaccharide (LPS) was measured by flow cytometry. MIF, CD74 and TLR4 co-localization in the MKN-45 cell line was performed by the immunoprecipitation.
  
  
  RESULTS
  CD74, MIF and TLR4 were found to be expressed in gastric cancer and increased significantly in the advanced stage, and were also associated with lymph node metastasis. Correlation analysis revealed that CD74 was positively correlated with MIF (r = 0.2367, P < 0.01) and both proteins were also associated with TLR4 (r = 0.4414, r = 0.5001, respectively, P < 0.01). LPS can significantly promote MKN-45 cell proliferation (3.027 ± 0.388 vs 4.201 ± 0.092, P < 0.05), induce MIF production (54.333 ± 2.906 pg/mL vs 29.667 ± 3.180 pg/mL, P < 0.01) and cell surface expression of CD74 (75.6% ± 4.046% vs 9.4% ± 0.964%, P < 0.01) at LPS concentration of 1 μg/mL compared to medium control. Knockdown of CD74 or using anti-CD74 and MIF antagonist ISO-1 significantly reduced LPS-induced MKN-45 cell proliferation (4.201 ± 0.092 vs 3.337 ± 0.087, 4.534 ± 0.222 vs 3.368 ± 0.290, 4.058 ± 0.292 vs 2.934 ± 0.197, respectively, P < 0.01). MIF, CD74 and TLR4 could co-localize in the MKN-45 cell line.
  
  
  CONCLUSIONS
  Upregulation of MIF, CD74 and TLR4 are associated with increasing clinical stage and provide an opportunity as novel gastric cancer chemoprevention and/or treatment strategy.
authors.affiliation: Department of Laboratory Medicine, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.
authors.forename: Ying-Xia
authors.initials: YX
authors.lastname: Zheng
authors.forename: Ming
authors.initials: M
authors.lastname: Yang
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Rong
authors.forename: Xiang-Liang
authors.initials: XL
authors.lastname: Yuan
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Ma
authors.forename: Zhi-Hao
authors.initials: ZH
authors.lastname: Wang
authors.forename: Li-Song
authors.initials: LS
authors.lastname: Shen
authors.forename: Long
authors.initials: L
authors.lastname: Cui
mesh_terms: D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007555:Isoxazoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008207:Lymphatic Metastasis; D008263:Macrophage Migration-Inhibitory Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D013274:Stomach Neoplasms / Q000235:genetics / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C462499:3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester; D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D007555:Isoxazoles; D008070:Lipopolysaccharides; D008263:Macrophage Migration-Inhibitory Factors; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C045084:invariant chain; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human