Abstract P5-11-10: Effect of claudin-3 protein expression on its sub-cellular localization and cellular motility in breast cancer cell lines
Authors: HM Petty, JM King, ME Cuevas, RA Sheller, MC Todd
Journal: Cancer Research (2013)

Abstract Tight Junctions (TJs), found at the apical side of vertebrate epithelial and endothelial junctions, serve as a selective barrier for the paracellular transport of solutes. Deregulation of these TJs has been shown to lead to loss of cell-to-cell adhesion. Subsequently, these cell barriers have become of interest in the study of cancer progression. Of the many proteins that make up a TJ, recent studies suggest a critical role for the claudin family in TJ formation and maintenance. Within this family of transmembrane proteins, we have previously shown claudin-3 to be overexpressed in two breast cancer cell lines, MCF-7 and MDA-MB-415, both of which are derived from metastatic deposits. In contrast, MDA-MB-157 cells (also of metastatic origin) expressed a lower level of claudin-3 relative to human mammary epithelial cells (HMEC). Indirect immunofluorescence (IF) analysis indicated that claudin-3 was primarily located in the membrane of MCF-7 and MDA-MB-415 but we also detected lower levels intracellularly. Notably, claudin-3 was not localized to the membrane in MDA-MB-157 cells. Instead, only an intracellular signal was detected. To more accurately determine the intracellular location of claudin-3 in the MDA-MB-157 cells, the current study, used differential detergent cell fractionation to isolate 4 sub-cellular fractions: cytosolic, membrane, nuclear and cytoskeletal. Consistent with the IF data, our preliminary analysis does not reveal membrane-specific claudin-3 signal but does show a low level of claudin-3 localized in the cytoskeletal fraction of MDA-MB-157 cells. This finding is striking as it indicates that claudin-3 does not necessarily play a role in the TJs of these cells (as indicated by the absence of claudin-3 localization to the membrane) but may function in the cytoskeleton, perhaps as part of a cell signaling pathway. Our earlier observations of MCF-7 and MDA-MB-415 cell lines (overexpress claudin-3) also revealed relatively high levels of claudin-3 in the cytoskeletal fraction but unlike MDA-MB-157, the majority of the protein was localized, as expected, in the membrane fraction. Deregulation and delocalization of claudin-3 in cell lines of metastatic origin suggests a possible role for this critical TJ protein in cellular invasiveness. Thus, we have begun to evaluate the impact of different levels of claudin-3 on cell motility using wound healing assays. Preliminary findings in MCF-7 show that siRNA-mediated suppression of claudin-3 protein slowed the migration of cells relative to their non-transfected counterparts. These findings suggest that overexpression of claudin-3 in MCF-7 cells may play a role in disrupting TJ integrity and thus contribute to enhanced cellular motility - a key component of tumor progression. Thus, it is possible that the siRNA-mediated suppression of claudin-3 may prove an effective therapeutic approach for breast cancers that overexpress claudin-3. We are currently in the process of analyzing both non-transfected and claudin-3- specific siRNA-transfected MDA-MB-415 and MDA-MB-157 to ascertain the effects of claudin-3 expression level on cellular motility in breast cancer cell lines that show either overexpressed or underexpressed levels of endogenous claudin-3 protein. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-10.