Carcinogenesis Advance Access originally published online on July 8, 2006
Carcinogenesis 2007 28(2):246-258; doi:10.1093/carcin/bgl120
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DNA-methylation-dependent alterations of claudin-4 expression in human bladder carcinoma
1 CNRS UMR5203, INSERM U661, Université Montpellier I, Université Montpellier II, Institut de Génomique Fonctionnelle, Département d'Oncologie Cellulaire et Moléculaire, 141 Rue de la Cardonille Montpellier F-34094 Cedex 5, France
2 Ludwig Institute for Cancer Research, Parkville Vic 3050, Australia
3 Service d'Anatomo-pathologie, CHU Groupe Hospitalisation Carémeau Nîmes, France
4 Service d'Urologie, CHU Groupe Hospitalisation Carémeau Nîmes, France
5 Service d'Urologie, Clinique Mutualiste Beausoleil 34000 Montpellier, France
6 Service d'Urologie, Polyclinique Kennedy 30000 Nîmes, France
7 Present address: Service d'Urologie, Clinique Mutualiste St Etienne, France
8 Present address: UMR CNRS 5810, Faculté de Pharmacie Montpellier, France
*To whom correspondence and requests for reprints should be addressed at: Institut de Génomique Fonctionnelle, Faculté de Pharmacie, Laboratoire de Biochimie, Bâtiment E, 15 Avenue C. Flahault, 34093 Montpellier Cedex 5, France. Fax: +33 0 4 67 66 81 49; Email: frederic.hollande{at}univ-montp1.fr
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.
Abbreviations: CPE, Clostridium Perfringens Enterotoxin; Dnmt3a, DNA methyl transferase 3a; MSP, methylation-specific PCR; SMA, smooth muscle actin; TER, Transepithelial resistance; TJ, tight junction
These authors contributed equally to the present work. Received March 11, 2006; revised June 4, 2006; accepted June 30, 2006.
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