Mechanistic Investigation Of Cellular Senescence Induced By Cathepsin D Knockdown In HeLa Cells

Cathepsin D (Cath D) is a ubiquitous protease that resides within lysosomes. It is one of the major players in cellular and tissue homeostasis. Cath D’s proteolytic activity is optimal in acidic environment such as lysosome, and is required for many proteins (such as procathespins and fibronectin) to execute their biological functions. In addition, once release into cytosol matrix, Cath D could induce apoptosis through proteolytic activation of pro-apoptotic pathways. In recent years, interest in Cath D’s role in tumor initiation and progression has been growing:Cath D is frequently over-expressed in tumor tissue compared with tumor adjacent or normal tissue, and the over-expression of Cath D could significantly enhance the invasiveness of cancer cells. Although numerous research has reported some correlation between Cath D and tumor pathogenesis, few of them illustrated the mechanistic links between Cath D and tumorigenesis. We speculated that the lack of mechanistic links mainly arose from two aspects:1) To investigate the function of Cath D in cancer cells, most of studies empolyed the cell model in which Cath D is over-expressed. Because the protein abundance of Cath D is often higher in cancer cells than in normal cells, the over-expression of Cath D is not likely to induce fundamental differences on either cells’ phenotype or signaling pathways.2) Most of Cath D studies only examined molecular changes within several specific proteins or singaling pathways, and there is insufficient study of Cath D’s global effect on molecular changes. As such, we propose that the systematic investigation of the proteomic changes brought about by down-regulating cathepsin D in cancer cells would deepen our understanding of Cath D’s function in cancer cells.The previous efforts of our lab in construction of stable HeLa cells with Cath D knockdown (HeLa-CR) and in phenotypic characterization had led to the finding that lowering Cath D abundance significantly induces cell senescence and cell cycle arrest in G2-M phase in HeLa cells. To globally and multi-dimensionally examine the proteomic changes, we used SILAC ({Stable Isotope Labeling with Amino Acid) technique to quantitatively compare subcellular proteomic changes in cytosol, lysosomal and nuclear fractions brought about by Cath D knockdown. Among many significant protein abundance changes, dozens of lysosome-residing proteins showed decreased abundance in lysosome and increased abundance in cytosol, indicating the status of lysosomal membrane integrity might be adversely affected. In addition, many proteins participated in oxidation reduction processes were down-regulated in all the three subcellular fractions, suggesting the oxidation reduction homeostasis might be disturbed.Based on quantitative proteomic data and the significance of cell homeostasis in senescence, we proposed that Cath D knockdown would lead to increased lysosomal membrane permeability (LMP) and reactive oxygen species (ROS) level, which mediate cell senescence. Subsequent analysis validated our hypothesis and established a causal role for oxdative stress assaults in mediating cell senescence induced by Cath D knockdown. In addition, we found that transcriptional activity of NRF2, an antioxidant protein master regulator, was down-regulated in spite of increased ROS. We revealed possible reasons behind this controversy through experiments:sustained oxidative stress might inhibit the transcriptional activity of NRF2 and anti-oxidant protein abundance. Our study suggested a novel role of Cath D in tumor initiation and progression, that is the maintenance of tissue homeostasis and hence the prevention of cell senescence during tumorigenesis.