The Study of WT1 and Cathepsin D in Breast Cancer Cells

Abstract

Wilms' tumor 1 (WT1) is a zinc finger transcription factor that involved in both biological process and pathological condition including cancer. Overexpression of WT1 was reported in leukemia and a variety of solid tumor such as lung cancer, ovarian cancer as well as breast cancer. The effect of WT1 function result in activation or repression role that depended on its isoform, cell type and partner proteins. In MCF- 7, ER-positive breast cancer cell line, WT1-related proteins were observed by knockdowned WT1 with siRNA technique and analyzed protein patterns with mass spectrometry. This experiment showed upregulation of cathepsin D (Cath D) when WT1 was downregulated. Cath D is aspartic protease presented mature enzyme in lysosome. It has a role in metabolic degradation of intracellular proteins, tissue homeostasis and activation hormone and precursors in the cell including regulation of cell growth and death program. Various studies have been found Cath D overexpression and proposed as a poor prognostic factor in breast cancer. Although Cath D expression was regulated by estrogen stimulation, but in TNBC which lacking of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression could also express Cath D expression. However, the relationship between WT1 and Cath D in TNBC has never been reported. In this study, the relationship of WT1 and Cath D protein was examined in breast cancer cell lines. Initially, endogenouse protein expression of WT1 and Cath D was observed in MCF-7 ER-positive cell line and TNBC cell lines including MDA-MB-468, MDA-MB-231 and Hs578T. The immunoblotting result showed WT1 and Cath D endogenouse protein expression was different between the groups of cell lines. WT1 protein expression was higher than Cath D in TNBC cell lines while Cath D expression was higher in ER- positive cell line. MDA-MB-468 TNBC cell which showed highest WT1 expression and lowest level of Cath D protein was chosen to clarify WT1-Cath D protein binding by immunoprecipitation (IP) technique. The IP result revealed that WT1 protein could form complex with Cath D in MDA-MB-468. Molecular docking also confirmed that WT1 could directly bind to Cath D. From WT1-Cath D docking structure, active sites of Cath D enzyme were not presented to WT1 cleavage sites. This result could suggest that WT1 interacted with Cath D with catalytic independent. However, WT1-Cath D binding was predicted at pH 7.5 that presented in cytosol and nucleus of the cell. At pH 5 or acidic compartment where presented in lysosome was further observed by PDB2PQR server for charge prediction. The result showed both pH 7.5 and 5 were not affected to WT1-Cath D interaction because the charge alteration was not presented on WT1 and Cath D interacted surface. Thus, WT1-Cath D binding might find at intracellular compartment where showed above pH.