Abstract
Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated ÎČ-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3ÎČ phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.
Highlights
Cysteine cathepsins perform various functions, including the processing of proteins during antigen presentation, bone modeling and epidermal homeostasis.[1]
In SW480 colon cancer and MDA-MB-231 breast cancer cells, CST1 knockdown using the pLenti expression system containing a short hairpin RNA for CST1 induced striking morphologic changes, including an enlarged nucleus and flattened cytoplasm, and increased SA-ÎČ-gal activity compared with pLenti-mock control cells (Figure 1b, upper panels)
We found that SA-ÎČ-gal activity in shCST1-MDA-MB-231 cells was inhibited by supplementation with conditioned medium obtained from MCF-7 cells stably expressing CST1, but not by supplementation with wild-type MCF-7 conditioned medium (Figure 2b)
Summary
Cysteine cathepsins perform various functions, including the processing of proteins during antigen presentation, bone modeling and epidermal homeostasis.[1]. The underlying relationship between CST1 and CatB, and their roles in tumor development, remains poorly understood. We observed that CST1 knockdown induced cellular senescence and decreased extracellular CatB activity in MDA-MB-231 and SW480 cancer cell lines. We hypothesized that the relationship between CST1 and CatB contributed to tumorigenesis via the prevention of cellular senescence. We further investigated the molecular mechanisms underlying CST1-mediated prevention against cellular senescence in cancer cells. Senescence under the indicated condition was quantified as the percentage of SA-ÎČ-gal-positive cells (lower). RNAs were extracted from cells, and real-time PCR analysis was performed to quantify the transcripts of CST1, IL-6 and chemokine ligand 20 (CCL20). Cells were seeded on to six-well plates (5 Ă cells per well) and cultured for the indicated number of days. One-way ANOVA was used for statistical analysis (*Po0.05). (g) Protein expression of p-Rb, total Rb, p21, cyclin D1 or CST1 was evaluated by western blotting. *Po0.05; **Po0.01; ***Po0.001 by Studentâs t-test
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