title: Lowering Endogenous Cathepsin D Abundance Results in Reactive Oxygen Species Accumulation and Cell Senescence.
abstract: Cathepsin D is reportedly to be closely associated with tumor development, migration, and invasion, but its pathological mechanism is not fully elucidated. We aimed to evaluate phenotypic changes and molecular events in response to cathepsin D knockdown. Lowering endogenous cathepsin D abundance (CR) induced senescence in HeLa cells, leading to reduced rate of cell proliferation and impaired tumorigenesis in a mouse model. Quantitative proteomics revealed that compared with control cells (EV), the abundances of several typical lysosomal proteases were decreased in the lysosomal fraction in CR cells. We further showed that cathepsin D knockdown caused increased permeability of lysosomal membrane and reactive oxygen species accumulation in CR cells, and the scavenging of reactive oxygen species by antioxidant was able to rescue cell senescence. Despite the increased reactive oxygen species, the proteomic data suggested a global reduction of redox-related proteins in CR cells. Subsequent analysis indicated that the transcriptional activity of nuclear factor erythroid-related factor 2 (Nrf2), which regulates the expression of groups of antioxidant enzymes, was down-regulated by cathepsin D knockdown. Importantly, Nrf2 overexpression significantly reduced cell senescence. Although transient oxidative stress promoted the accumulation of Nrf2 in the nucleus, we showed that the Nrf2 protein exited nucleus if oxidative stress persisted. In addition, when cathepsin D was transiently knocked down, the cathepsin-related events followed a sequential order, including lysosomal leakage during the early stage, followed by oxidative stress augmentation, and ultimately Nrf2 down-regulation and senescence. Our results suggest the roles of cathepsin D in cancer cells in maintaining lysosomal integrity, redox balance, and Nrf2 activity, thus promoting tumorigenesis. The MS Data are available via ProteomeXchange with identifier PXD002844.
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.|§University of Chinese Academy of Sciences, Beijing, China, 100049.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Su
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhu
authors.affiliation: ¶Proteomics Division, BGI-Shenzhen, Shenzhen, Guangdong, China, 518083.
authors.forename: Liang
authors.initials: L
authors.lastname: Lin
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.|§University of Chinese Academy of Sciences, Beijing, China, 100049.
authors.forename: Xianwei
authors.initials: X
authors.lastname: Chen
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.|§University of Chinese Academy of Sciences, Beijing, China, 100049.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: ¶Proteomics Division, BGI-Shenzhen, Shenzhen, Guangdong, China, 518083.
authors.forename: Jin
authors.initials: J
authors.lastname: Zi
authors.affiliation: ‖Beijing Protein Innovation, Beijing, China, 101318.
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Dong
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.|§University of Chinese Academy of Sciences, Beijing, China, 100049.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Xie
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.|§University of Chinese Academy of Sciences, Beijing, China, 100049.
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.
authors.forename: Ju
authors.initials: J
authors.lastname: Zhang
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Zhu
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101.
authors.forename: Dan
authors.initials: D
authors.lastname: Xu
authors.affiliation: **Laboratory of Cell and Molecular Biology, Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, 100021.
authors.forename: Ningzhi
authors.initials: N
authors.lastname: Xu
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101; siqiliu@big.ac.cn louxm@big.ac.cn.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Lou
authors.affiliation: From the ‡CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 100101; siqiliu@big.ac.cn louxm@big.ac.cn.|§University of Chinese Academy of Sciences, Beijing, China, 100049.
authors.forename: Siqi
authors.initials: S
authors.lastname: Liu
mesh_terms: D000072283:A549 Cells; D000818:Animals; D002402:Cathepsin D / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D016922:Cellular Senescence; D015536:Down-Regulation; D005260:Female; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D040901:Proteomics / Q000379:methods*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D017382:Reactive Oxygen Species; C533922:CTSD protein, human; D002402:Cathepsin D