Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor

ABSTRACT The epithelial mesenchymal transition (EMT) promotes tumor migration and invasion by downregulating epithelial markers such as E-cadherin and upregulating mesenchymal markers such as vimentin. Cathepsin L (Cat L) is a cysteine protease that can proteolytically activate CCAAT displacement protein/cut homeobox transcription factor (CUX1). We hypothesized that nuclear Cat L may promote EMT via CUX1 and that this could be antagonized with the Cat L-specific inhibitor Z-FY-CHO. Mesenchymal prostate (ARCaP-M and ARCaP-E overexpressing Snail) and breast (MDA-MB-468, MDA-MB-231, and MCF-7 overexpressing Snail) cancer cells expressed lower E-cadherin activity, higher Snail, vimentin, and Cat L activity, and a p110/p90 active CUX1 form, compared to epithelial prostate (ARCaP-E and ARCaP-Neo) and breast (MCF-7 and MCF-7 Neo) cancer cells. There was increased binding of CUX1 to Snail and the E-cadherin promoter in mesenchymal cells compared to epithelial prostate and breast cells. Treatment of mesenchymal cells with the Cat L inhibitor Z-FY-CHO led to nuclear-to-cytoplasmic relocalization of Cat L, decreased binding of CUX1 to Snail and the E-cadherin promoter, reversed EMT, and decreased cell migration/invasion. Overall, our novel data suggest that a positive feedback loop between Snail-nuclear Cat L-CUX1 drives EMT, which can be antagonized by Z-FY-CHO. Therefore, Z-FY-CHO may be an important therapeutic tool to antagonize EMT and cancer progression.

Anaplasma phagocytophilum Increases Cathepsin L Activity, Thereby Globally Influencing Neutrophil Function

ABSTRACT Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, is an unusual obligate intracellular pathogen that persists in neutrophils. A. phagocytophilum increases the binding of a repressor, CCAAT displacement protein (CDP), to the gp91phox promoter, thereby diminishing the host oxidative burst. We now show that A. phagocytophilum infection also enhances the binding of CDP to the promoters of human neutrophil peptide 1 and C/EBPε—molecules important for neutrophil defense and maturation—suggesting that this is a general strategy used by this pathogen to alter polymorphonuclear leukocyte function. To explore the mechanism by which A. phagocytophilum increases CDP activity, we assessed the effects of this microbe on cathepsin L, a protease that cleaves CDP into a form with increased DNA binding ability. A. phagocytophilum infection resulted in elevated cathepsin L activity and the proteolysis of CDP. Blocking the action of cathepsin L with a chemical inhibitor or small interfering RNA targeting of this molecule caused a marked reduction in the degree of A. phagocytophilum infection. These data demonstrate that increasing cathepsin L activity is a strategy used by A. phagocytophilum to alter CDP activity and thereby globally influence neutrophil function. As therapeutic options for A. phagocytophilum and related organisms are limited, these results also identify a cellular pathway that may be targeted for the treatment of A. phagocytophilum infection.