Abstract B93: Nuclear cathepsin L (cat L) is associated with epithelial mesenchymal transition (emt) in prostate cancer cells

Prostate cancer (PCa) is the most frequently diagnosed cancer in men, the second leading cause of male cancer deaths in the U.S, and also presents the greatest racial disparity of any cancer in the U.S. The incidence and mortality of PCa is higher in African-American men (AA), compared to other ethnic groups. 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 promotes EMT by using its zinc finger domains to repress epithelial-associated target genes such as E-cadherin and occludin. 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 when Cat L is localized in the nucleus. CDP/CUX can be proteolytically activated by Cat L from p200 inactive form to p110 and p90 active forms, and subsequently repress E-cadherin and activate Snail by binding to their promoter regions. We recently published that Snail could up-regulate Cat L expression and activity in prostate cancer cells. We hypothesized that nuclear Cat L may promote EMT and prostate cancer progression and that antagonizing Cat L activity would lead to Mesenchymal Epithelial Transition (MET). We stained African American and Caucasian human normal and prostate cancer tissue from the Fox Chase Cancer Center for Snail and Cat L to examine expression. We utilized prostate cancer cell lines and EMT models to analyze EMT markers and Cat L expression by western blot analysis, Q-PCR and immunofluorescence. We also examined Cat L activity using gelatin zymography and subsequently treated mesenchymal cells with 1 µM, 5 µM or 20 µM Z-FY-CHO (Cat L specific inhibitor) for 72 hours. We also performed invasion and migration assays. Staining of human prostate patient tissue revealed that Snail and Cat L levels were lower in normal tissue vs cancer tissue and nuclear localization of Cat L appeared in earlier stages of prostate cancer in African American tissue compared to that of Caucasian cancer tissue. Interestingly, analysis of epithelial (ARCaP-E) and mesenchymal (ARCaP-M or ARCaP-E overexpressing Snail) prostate cancer cells revealed that mesenchymal cells displaying higher Snail expression showed increased Cat L expression and activity, in addition to Cat L being localized in the cytoplasm and nucleus, whereas Cat L was predominantly cytoplasmic in epithelial cells with low Snail expression. Additionally, the African American cell lines (E006-AA and E006-AA ht) displayed high Snail and Cat L expression/activity compared to LNCaP cells. Mesenchymal cells expressed higher p110 and p90 active form of CDP/CUX than epithelial cells that expressed the p200 inactive form. Additionally, Snail overexpression increased p110/90 CDP/CUX in the nuclear fraction of ARCaP-E overexpressing Snail cells. Treatment of mesenchymal cells with Cat L inhibitor, Z-FY-CHO resulted in decreased vimentin and Cat L expression, while E-cadherin increased, suggestive of MET. This was associated with decreased cell migration and invasion. We further observed that treatment with Z-FY-CHO changes the localization of Cat L from nuclear to cytoplasmic. Overall, our novel data suggests that Snail may promote nuclear Cat L that may be associated with proteolytic degradation of CDP/CUX and EMT, and the inhibition of Cat L could play a role in reverting EMT and preventing tumor progression. Citation Format: Liza J. Burton, Jodi Dougan, Valerie Odero-Marah. Nuclear cathepsin L (cat L) is associated with epithelial mesenchymal transition (emt) in prostate cancer cells. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B93.