Interleukin-18 enhances breast cancer cell migration via down-regulation of claudin-12 and induction of the p38 MAPK pathway.

Interleukin-18 (IL-18) was recently reported to have a pro-tumor effect in various cancers. Increased IL-18 levels in the serum of cancer patients correlated with malignancy, and IL-18 acts a crucial factor for cell migration in gastric cancer and melanoma. Claudins, which are the most important tight junction proteins, are also linked with cancer progression and metastasis. However, the relationship between claudins and IL-18 is not well-understood. Here, we show that the migratory ability of MCF-7 cells was reduced when endogenous IL-18 expression was inhibited with IL-18 siRNA. Moreover, exogenous IL-18 enhanced breast cancer cell migration and suppressed the expression of the tight junction proteins claudin-1, claudin-3, claudin-4, and claudin-12 in MCF-7 cells. Knockdown of claudin-3, claudin-4, and claudin-12, but not claudin-1, increased breast cancer migration with maximal effects observed in claudin-12 siRNA-transfected cells. To investigate whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in IL-18-induced cell migration and claudin-12 expression, cells were pretreated with SB203580 (an inhibitor of p38 MAPK) or PD98059 (an inhibitor of ERK1/2) prior to the addition of IL-18. Although pretreatment of MCF-7 cells with SB203580 blocked both the enhanced cell migration and the decreased claudin-12 expression, PD98059 only blocked cell migration and did not affect claudin-12 expression. In addition, exogenous IL-18 induced rapid phosphorylation of p38 MAPK. These results suggest that IL-18 is an important factor inducing breast cancer cell migration through down-regulation of claudin-12 and activation of the p38 MAPK pathway.

Antitumor effects of heparin-polyethyleneimine nanogels delivering claudin-3-targeted short hairpin RNA combined with low-dose cisplatin on ovarian cancer.

Cisplatin is normally administered in chemotherapy for ovarian cancer, but is accompanied by severe dose-dependent toxicity. The combination of cisplatin with other antitumor agents may be a useful alternative for achieving higher antitumor efficiency and lower toxicity. Claudin-3 (CLDN3), a commonly upregulated gene in 90% of ovarian cancers, has been identified as a novel therapeutic target of ovarian cancer. Therefore, in the present study, we constructed a recombinant plasmid carrying an shRNA targeting CLDN3 (pshCLDN3), and investigated the antitumor effects of the combination therapy of pshCLDN3 and a low-dose of cisplatin for the treatment of ovarian cancer. Heparin-polyethyleneimine (HPEI) nanogel, a novel gene carrier with superior biodegradability, excellent blood compatibility and low-toxicity, was used to deliver pshCLDN3 into ovarian cancer cells. The knockdown efficiency was determined by western blot analysis and CLDN3 immunostaining. Nude mice bearing intraperitoneal ovarian carcinomas were treated with pshCLDN3/HPEI complexes, low-dose cisplatin, pshCLDN3/HPEI plus low-dose cisplatin or control agents, respectively. The results showed that pshCLDN3/HPEI effectively suppressed the expression of CLDN3 in ovarian cancer. The combination therapy of pshCLDN3/HPEI and low-dose cisplatin exhibited enhanced antitumor activity, when compared with either agent alone, as evidenced by mean tumor weight analysis, Ki-67 immunostaining analysis and TUNEL assay, without obvious systemic toxicity. These results indicate that pshCLDN3/HPEI combined with low-dose cisplatin demonstrates apparent synergistic antitumor activity without marked toxicity. Our study offers a novel therapeutic strategy for the treatment of ovarian cancer.

Regulation of the Epithelial-Mesenchymal Transition by Claudin-3 and Claudin-4.

The mechanisms that control intracellular adhesion are central to the process of invasion and metastasis. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are major structural molecules of the tight junctions that link epithelial cells. Our prior work has demonstrated that knockdown of the expression of either CLDN3 or CLDN4 produces marked changes in the phenotype of ovarian carcinoma cells including increases in growth rate in vivo, migration, invasion, metastasis, and drug resistance, similar to those produced by the epithelial-to-mesenchymal transition (EMT). We postulated that these changes may result from the ability of CLDN3 or CLDN4 to suppress EMT. In this study we found that knockdown of either CLDN3 or CLDN4 increased cell size and resulted in flattened morphology. While knockdown of CLDN3 or CLDN4 did not alter the expression of vimentin, it significantly down-regulated the level of E-cadherin and up-regulated N-cadherin expression. Conversely, over-expression of CLDN3 or CLDN4 in a cell line that does not express endogenous CLDN3 or CLDN4 decreased N-cadherin expression. Re-expression of E-cadherin in the CLDN3 or CLDN4 knockdown cells reduced migration, invasion and tumor growth in vivo. Loss of either CLDN3 or CLDN4 resulted in activation of the PI3K pathway as evidenced by increased Akt phosphorylation, elevated cellular PIP3 content and PI3K activity as well as up-regulation of the mRNA and protein levels of the transcription factor Twist. Taken together, these findings suggest that CLDN3 and CLDN4 function to sustain an epithelial phenotype and that their loss promotes EMT.

Tight junction proteins claudin-3 and claudin-4 control tumor growth and metastases.

The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), and activation of ß-catenin pathway signaling as evidenced by increases in nuclear ß-catenin, the dephosphorylated form of the protein, and transcriptional activity of ß-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting ß-catenin signaling.