title: Metastasis to Bone in Human Cancer Is Associated with Loss of Occludin Expression.
abstract: BACKGROUND
  Occludin is an integral membrane protein localised at tight junctions (TJ). There is no consensus regarding its paramount role in TJ. In previous work we showed that occludin is aberrantly expressed in both human breast tissues and cancer cell lines. This study demonstrates a link to bone metastasis in human cancer.
  
  
  METHODS
  Primary breast tumours (n=124) and matched normal tissues (n=30) were processed for quantitative polymerase chain reaction (QPCR) analysis. A hammerhead ribozyme was constructed to create occludin knockdown cell lines, MDA-MB-231(ΔOcc) and PC-3(ΔOcc). The effect of human bone matrix extract (BME) was investigated using cell growth and electric cell impedance sensing (ECIS) technology to measure changes in attachment/migration. Trans-epithelial resistance (TER) was measured for assessing changes in TJ function. Cells used were MDA-MB-231, PC-3, CORL-23, SKMES-1 and A-549 human cancer cell lines.
  
  
  RESULTS
  Tumours from patients with bone metastasis had significantly lower occludin expression compared to those remaining alive/well (60.7±21 vs. 331±98, respectively, p=0.008). This was striking in ductal carcinomas, where patients alive/well had significantly higher occludin expression compared to those with bone metastasis (391±12.5 vs. 67.9±28, respectively, p=0.0014). ECIS demonstrated that MDA-MB-231(ΔOcc) showed reduced attachment to 5% BME compared to controls (84% vs. 100%) that prevented closure of wounded cell layers. Moreover, these cells had reduced growth on BME. In addition, BME changed the TER of a number of human cell lines and was able to effect changes in the growth of MDA-MB-241 and PC-3 cells, with greater effect on knockdown cells.
  
  
  CONCLUSIONS
  This is the first study to demonstrate that occludin expression has a clear relationship with bone metastasis in human cancer. The discrepancy between this and the in vitro data indicating a reduction in migration/growth rate of occludin knockdown indicates that loss of occludin leads to complex changes in human cancer cell phenotype.
authors.affiliation: Cardiff-China Medical Research Collaborative, School of Medicine, Cardiff University, Cardiff, U.K. martinta1@cf.ac.uk.
authors.forename: Tracey A
authors.initials: TA
authors.lastname: Martin
authors.affiliation: Cardiff-China Medical Research Collaborative, School of Medicine, Cardiff University, Cardiff, U.K.
authors.forename: Nicola
authors.initials: N
authors.lastname: Jordan
authors.affiliation: Cardiff-China Medical Research Collaborative, School of Medicine, Cardiff University, Cardiff, U.K.
authors.forename: Eleri L
authors.initials: EL
authors.lastname: Davies
authors.affiliation: Cardiff-China Medical Research Collaborative, School of Medicine, Cardiff University, Cardiff, U.K.
authors.forename: Wen G
authors.initials: WG
authors.lastname: Jiang
mesh_terms: D001857:Bone Matrix / Q000378:metabolism; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000401:mortality / Q000556:secondary*; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D015536:Down-Regulation; D017097:Electric Impedance; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D008297:Male; D062793:Occludin / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016019:Survival Analysis; D019108:Tight Junctions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: C568985:OCLN protein, human; D062793:Occludin; D016337:RNA, Catalytic; C427204:hammerhead ribozyme
keywords: HGF; breast cancer; metastasis; occludin; progression; tight junction