title: DNA-methylation-dependent alterations of claudin-4 expression in human bladder carcinoma.
abstract: The expression pattern of tight junction (TJ) proteins is frequently disrupted in epithelial tumors. In particular, isoform- and organ-specific alterations of claudins have been detected in human cancers, highlighting them as interesting tools for the prognosis or treatment of various carcinomas. However, the molecular mechanisms responsible for these alterations are seldom identified. Here, we analyzed the expression and localization of claudins 1, 4, and 7 in human bladder carcinoma. Claudin-4 expression was significantly altered in 26/39 tumors, contrasting with the rare modifications detected in the expression of claudins 1 and 7. Overexpression of claudin-4 in differentiated carcinomas was followed by a strong downregulation in invasive/high-grade tumors, and this expression pattern was associated to the 1-year survival of bladder tumor patients. A CpG island was identified within the coding sequence of the CLDN4 gene, and treatment with a methyl-transferase inhibitor restored expression of the protein in primary cultures prepared from high-grade human bladder tumors. In addition, claudin-4 expression correlated with its gene methylation profile in healthy and tumoral bladders from 20 patients, and downregulation of claudin-4 expression was detected in the urothelium of mice overexpressing DNA methyl transferase 3a (Dnmt3a). Delocalization of claudins 1 and 4 from TJs was observed in most human bladder tumors and in the bladder tumor cell line HT-1376. Although the CLDN4 gene was unmethylated in these cells, pharmacological inhibition of methyl transferases re-addressed the two proteins to TJs, resulting in an increase of cell polarization and transepithelial resistance. These biological effects were prevented by expression of claudin-4-specific siRNAs, demonstrating the important role played by claudin-4 in maintaining a functional regulation of homeostasis in urothelial cells. Results of this study indicate that the TJ barrier is disrupted from early stages of urothelial tumorigenesis. In addition, we identified hypermethylation as the mechanism leading to the alteration of claudin-4 expression, and maybe also localization, in bladder carcinoma.
authors.affiliation: CNRS UMR5203, INSERM U661, Université Montpellier I, and Service d'Anatomo-pathologie, CHU Groupe Hospitalisation Carémeau, Nîmes, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Boireau
authors.forename: Michael
authors.initials: M
authors.lastname: Buchert
authors.forename: Michael S
authors.initials: MS
authors.lastname: Samuel
authors.forename: Julie
authors.initials: J
authors.lastname: Pannequin
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Ryan
authors.forename: Armelle
authors.initials: A
authors.lastname: Choquet
authors.forename: Héliette
authors.initials: H
authors.lastname: Chapuis
authors.forename: Xavier
authors.initials: X
authors.lastname: Rebillard
authors.forename: Christophe
authors.initials: C
authors.lastname: Avancès
authors.forename: Matthias
authors.initials: M
authors.lastname: Ernst
authors.forename: Dominique
authors.initials: D
authors.lastname: Joubert
authors.forename: Nicolas
authors.initials: N
authors.lastname: Mottet
authors.forename: Frédéric
authors.initials: F
authors.lastname: Hollande
mesh_terms: D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D062506:Claudin-4; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D015536:Down-Regulation; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D034741:RNA, Small Interfering; D001749:Urinary Bladder Neoplasms / Q000378:metabolism* / Q000473:pathology
chemical_list: C568157:CLDN4 protein, human; D062506:Claudin-4; C568158:Cldn4 protein, mouse; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A