Abstract
BackgroundClaudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. The objective of this study was to determine the functional role that claudin-4 plays in apoptosis resistance and migration as well as the therapeutic utility of targeting claudin-4 activity with a small mimic peptide.MethodsWe examined claudin-4 activity in human ovarian tumor cell lines (SKOV3, OVCAR3, PEO4) using in vitro caspase and scratch assays as well as an in vivo mouse model of ovarian cancer. Claudin-4 activity was disrupted by treating cells with a small peptide that mimics the DFYNP sequence in the second extracellular loop of claudin-4. Claudin-4 expression was also altered using shRNA-mediated gene silencing.ResultsBoth the disruption of claudin-4 activity and the loss of claudin-4 expression significantly increased tumor cell caspase-3 activation (4 to 10-fold, respectively) in response to the apoptotic inducer staurosporine and reduced tumor cell migration by 50 %. The mimic peptide had no effect on cells that lacked claudin-4 expression. Female athymic nude mice bearing ZsGreen-PEO4 ovarian tumors showed a significant decrease in ovarian tumor burden, due to increased apoptosis, after treatment with intraperitoneal injections of 4 mg/kg mimic peptide every 48 h for three weeks, compared to control peptide treated mice.ConclusionClaudin-4 functionally contributes to both ovarian tumor cell apoptosis resistance and migration and targeting extracellular loop interactions of claudin-4 may have therapeutic implications for reducing ovarian tumor burden.
Highlights
Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile
Western blot analysis revealed that native SKOV3, OVCAR3, and PEO4 ovarian tumor cells expressed claudin-4
Western blot analysis confirmed that claudin-4 protein expression in claudin-4 shRNA-treated SKOV3 cells, “SKOV3_cld4KD”, were reduced compared to empty vector control shRNA-treated, “SKOV3_contKD” (Fig. 1c)
Summary
Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. Claudin-4 is a transmembrane protein that interacts with other claudin-4 proteins expressed at the surface of neighboring cells through extracellular loop interactions [1, 2] These interactions of claudin-4 have been most well defined in normal epithelia in culture, where claudin-4 is often restricted to tight junction structures and has been shown to provide a barrier to paracellular diffusion [3, 4]. Claudin-4 is highly expressed in ovarian tumor cell lines categorized in each of the five different molecular subtypes of epithelial ovarian cancer recently described by Tan and colleagues, which includes epithelial- [OVCA429, CaOV3], mesenchymal[PEO1, SKOV3], and stem-like [OVCAR3, OVCAR5] subtypes [16,17,18]. It is possible that the activity of claudin-4 in tumor cells may, stem from their function in the normal epithelium from which the tumor arises and this activity is enhanced due to loss of regulation
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