Cathepsin L Inhibition Reverts Epithelial Mesenchymal Transition In Prostate And Breast Cancer Cells

The Epithelial Mesenchymal Transition (EMT) is a key event in tumor migration and invasion processes whereby epithelial cells lose polarity together with cell-cell contacts associated with loss of epithelial markers such as E-cadherin and then undergo a dramatic remodeling of the cytoskeleton associated with expression of mesenchymal markers such as vimentin, as well as increased production of extracellular matrix (ECM)-degrading proteases. Snail is a gene that promotes epithelial mesenchymal transition (EMT) by using its zinc finger domains to bind to target genes that would otherwise stop EMT. Cathepsin L (Cat L) is a cysteine protease involved in cell invasion and has recently been associated with poor prognosis in Colon and Breast Cancer with Cat L being localized in the nucleus. We have shown previously that Snail-mediated EMT can be antagonized by muscadine grape skin extract (MSKE), a natural product rich in anthocyanin. In preliminary data we found that Snail, an inducer of EMT, could up-regulate Cat L activity and expression in breast cancer cells. We hypothesized that antagonizing Cat L activity would lead to mesenchymal epithelial transition (MET). We utilized breast and prostate EMT models to analyze Cat L activity using gelatin zymography and subsequently treated mesenchymal cells with 1μM, 5μM or 20μM Z-FY-CHO (Cat L specific inhibitor) or MSKE for 72 hours. We performed western blot analysis and immunofluorescence for EMT markers as well as invasion and migration assays. Our data indicates that mesenchymal MDA-MB-231 breast cancer cells showed higher Cat L expression and activity as compared to epithelial MCF-7 breast cancer cells. Interestingly, immunofluorescence analysis revealed cells displaying a higher snail expression have an increase in Cathepsin L activity and expression as well as Cat L being localized in the cytoplasm and nucleus. Additionally, mesenchymal breast (MDA-MB-231, MCF-7 cells overexpressing Snail) and prostate (ARCaP-M, ARCaP-E cells overexpressing Snail) cells displayed decreased expression of vimentin and Cat L expression and activity when treated with Z-FY-CHO or MSKE, while E-cadherin increased suggestive of MET. This was associated with decreased cell migration and invasion. We also show for the first time that treatment with MSKE or Z-FY-CHO changes the localization of Cat L from nuclear to cytoplasmic. Based on these results, the inhibition of Cat L could play a role in reverting EMT and preventing tumor progression. Citation Format: Liza J. Burton, Bethany Smith, Manu Platt, Valerie Marah. Cathepsin L inhibition reverts epithelial mesenchymal transition in prostate and breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2015-4105