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. 2019 May;26(5):812-825.
doi: 10.1038/s41418-018-0153-0. Epub 2018 Jul 13.

Regulating BRCA1 protein stability by cathepsin S-mediated ubiquitin degradation

SeoYoung Kim  1 , Hee Jin  1 , Hang-Rhan Seo  2 , Hae June Lee  3 , Yun-Sil Lee  4
Affiliations

Regulating BRCA1 protein stability by cathepsin S-mediated ubiquitin degradation

SeoYoung Kim et al. Cell Death Differ. 2019 May.

Abstract

Cathepsin S (CTSS) is a cysteine protease that is thought to play a role in many physiological and pathological processes including tumor growth, angiogenesis, and metastasis; it has been identified as a radiation response gene. Here, we examined the role of CTSS in regulating the DNA damage response in breast cancer cells. Activating CTSS (producing the cleavage form of the protein) by radiation induced proteolytic degradation of BRCA1, which ultimately suppressed DNA double-strand break repair activity. Depletion of CTSS by RNAi or expression of a mutant type of CTSS enhanced the protein stability of BRCA1 by inhibiting its ubiquitination. CTSS interacted with the BRCT domain of BRCA1 and facilitated ubiquitin-mediated proteolytic degradation of BRCA1, which was tightly associated with decreased BRCA1-mediated DNA repair activity. Treatment with a pharmacological CTSS inhibitor inhibited proteolytic degradation of BRCA1 and restored BRCA1 function. Depletion of CTSS by shRNA delayed tumor growth in a xenograft mouse model, only in the presence of functional BRCA1. Spontaneously uced rat mammary tumors and human breast cancer tissues with high levels of CTSS expression showed low BRCA1 expression. From these data, we suggest that CTSS inhibition is a good strategy for functional restoration of BRCA1 in breast cancers with reduced BRCA1 protein stability.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
CTSS modulates BRCA1 protein expression. a CTSS protease activity was detected using MCF7 cells after exposure to 10 Gy (IR) at indicated time points. *p < 0.05 vs. untreated control cells (Student’s t-test). b MCF7 cells were irradiated with 1 Gy and 10 Gy and after indicated time points, Western blotting was performed on the cells. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control. c Western blot analysis of CRISPR-Cas9-Control (Con) and -CTSS KO MCF7 cells in the presence or absence of 10 Gy radiation. d MCF7 cells transfected with WT-CTSS, si-RNA, or mutant type of CTSS (C25A; active site Cys25 mutated to Ala). e Western blotting of cytosolic and nuclear fractions from MCF7 cells was performed. Fraction purity and equal loading were assessed by Western blots for Lamin B and GAPDH. f MCF7 cells were irradiated (10 Gy) for 2 h and stained with Flag antibody for BRCA1 (green, g), V5 antibody for CTSS (red, R), and DAPI. Cells were analyzed using immunofluorescence microscopy. Quantification of BRCA1 staining was performed by dividing into V5 positive and negative MCF7 cells. The error bars represent S.D. Data are expressed as the fold change relative to the control. *p < 0.05 (Student’s t-test)
Fig. 2
Fig. 2
CTSS interacts with the BRCT domain of BRCA1 and inhibits BRCA1 protein expression. a Constructs of V5-CTSS and V5-C25A (active site of Cys25 mutated to Ala) were transiently transfected into Flag-BRCA1 transfected MCF7 cells, and cell extracts were subjected to immunoprecipitation (IP) and immunoblotting (IB). b Schematic presentation of BRCA1 domain, including RING, Rad51 interacting, and BRCT domains (upper). MCF7 cells were transfected with BRCA1 fragments (F1 to F6) encoding Myc with or without full-length V5-CTSS and analyzed by Western blotting (bottom). c Constructs of V5-CTSS and V5-C25A were transiently transfected into Flag-BRCT transfected MCF7 cells, and cell extracts were subjected to immunoprecipitation (IP) and immunoblotting (IB). d Schematic structure of BRCA1, BRCT, and BRCT deletion construct (ΔBRCT). MCF7 cells were transfected with WT-BRCA, BRCT with and without WT-CTSS, C25A and analyzed by Western blotting. e MCF7 cells were transfected with WT-BRCA and ΔBRCT constructs with and without WT-CTSS or sh-CTSS and analyzed by Western blotting. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control
Fig. 3
Fig. 3
CTSS increases ubiquitin-mediated degradation of BRCA1. a Western blot analysis in control or si-CTSS transfected MCF7 cells treated with 100 μg/ml cycloheximide (CHX) for various lengths of time. b MCF7 cells were transfected with WT-BRCA1 or BRCT deletion mutant of BRCA1 (ΔBRCT) and incubated in the presence of CHX, and Western blotting was performed. Band density was expressed as the fold change relative to the control (Mean ± SD of 3 experiments). c, d For ubiquitination assays, MCF7 cells were transfected with control, WT-CTSS, C25A-CTSS, sh-CTSS, or BRCA1 constructs after transfection with ubiqutine construct (Ub). Cell lysates were immunoprecipitated (IP) and immunoblotted (IB)
Fig. 4
Fig. 4
CTSS enhances BRCA1 downstream functions. a Promoter reporter construct gadd45 was co-transfected with BRCA1, ΔBRCT, CTSS, si-Con, si-BRCA1, or si-CTSS into MCF7 cells. Cells were collected and subjected to luciferase assay. b MCF7 (upper), MDA-MB-231 (middle) and MDA-MB-436 (bottom) cells were co-transfected with WT-CTSS or si-CTSS either the si-Con or si-BRCA1 as indicated. c CTSS and BRCA1 protein expression following treatment of MCF7 or MDA-MB-231 cells with CTSS specific inhibitor VBY-036 (10 μM) (upper). Promoter activity gadd45 was following treatment of MCF7 or MDA-MB-231 cells with CTSS-specific inhibitor VBY-036 or si-CTSS (bottom). d MCF7 cells were treated with IR (10 Gy) after transfection with WT-BRCA1, WT-CTSS, si-CTSS, or si-BRCA1 constructs and Western blotting was performed. Protein levels were quantified using Image J software and data are expressed as the fold change relative to the negative control. Normalized luciferase activities were referred to the activity of extracts. Graphs represent Mean ± SD of three experiments. *p < 0.05 and **p < 0.01 (ANOVA)
Fig. 5
Fig. 5
RING domain of BRCA1 is not important for CTSS-mediated BRCA1 degradation. a Western blot analysis in WT-BRCA1 and RING domain deleted mutant (ΔRING) transfected MCF7 cells treated with 100 μg/ml cycloheximide (CHX) for various lengths of time. b MCF7 cells were transfected with WT-BRCA1 or ΔRING with or without siRNA of CTSS (si-CTSS) and incubated in the presence of CHX and Western blotting was performed. Band density was expressed as the fold change relative to the control (Mean ± SD of 3 experiments). c MCF7 cells were transfected with WT-BRCA1, ΔRING, or ΔBRCT. For ubiquitination assays, MG132 (10 μM) was treated after transfection of WT-CTSS, and cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with ubiqutin construct (Ub). d Promoter reporter construct gadd45 in MCF7 (left) and MDA-MB-231 (right) cells were co-transfected with WT-BRCA1, ΔRING, or ΔBRCT as indicated. Normalized luciferase activity referred to the activity of the extracts. Data are representative of three independent experiments with similar results. Graphs represent mean ± SD of three experiments. *p < 0.05 (ANOVA). e Cell lysates after transfection of sh-CTSS or treatment of VBY-036, a CTSS specific inhibitor at 10 μM were immunoprecipitated (IP) with cyclin B1 and immunoblotted (IB) with ubiqutin construct (Ub). Western blotting was also performed
Fig. 6
Fig. 6
CTSS decreases DNA damage responses. a Control or WT-CTSS was transiently transfected into MCF7 cells. The levels of BRCA1 and CTSS in the dsDNA pull-down lysates, as well as in complete whole nuclear extracts, were analyzed. b MCF7 cells were irradiated with 10 Gy IR after transfection with control or sh-CTSS, and Western blotting was performed at indicated time points. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control (left). Immunofluorescence analysis for γH2AX foci were performed after 10 Gy IR (right upper). Apoptotic cells after 10 Gy IR were evaluated by PI staining in CRISPR-Cas9 CTSS KO MCF7 cell lines. *p < 0.01 vs. corresponding control cells (ANOVA) (right bottom). c After 10 Gy radiation, Western blotting was performed at indicated time points with or without treatment of CTSS-specific inhibitor VBY-036 (10 μM). d The ratio of GFP + cells transfected with sh-CTSS, BRCA1, ΔBRCT, or ΔRING expression in MCF7 cells that stably expressed DR-GFP were analyzed by FACS. e GFP + cells after treatment of VBY-036 (10 μM) or sh-CTSS in MCF7 cells that stably expressed DR-GFP were analyzed by FACS (mean ± SD from 3 different experiments). *p < 0.05 (ANOVA)
Fig. 7
Fig. 7
Inhibition of IR-mediated cell death by CTSS. a MCF7 cells were transfected with a control or WT-BRCA1. Western blots of cleaved-PARP and BRCA1 expression were performed (upper). Cell death was evaluated by PI staining after 12, 24, and 48 h of 10 Gy IR (bottom). b MCF7 cells were transfected with si-CTSS or were treated with VBY-036 (10 μM). Western blots of cleaved-PARP and BRCA1 expression were performed (upper). Prevalence of cell death was evaluated by PI staining after 48 h of exposure to 10 Gy (bottom). c MDA-MB-436 cells with or without WT-BRCA1 were treated with si-CTSS or VBY-036 (10 μM). After 48 h of 10 Gy IR, Western blotting (left) or PI staining (right) was performed. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control. The graphs depict the mean ± SD of PI-positive cells. *p < 0.05 and **p < 0.01 (ANOVA)
Fig. 8
Fig. 8
Delayed tumor growth by inhibition of CTSS with recovery of BRCA1 expression. a Western blotting (total 8 tissues, upper) or immunohistochemistry (IHC) (total 4 tissues, bottom) for BRCA1 and CTSS was performed using rat mammary tumor tissues. b Western blotting was performed using MBA-MD-231 cells stably transfected of sh-CTSS, sh-BRCA1, and sh-CTSS/sh-BRCA1 (double deletion of CTSS/BRCA1) (upper). Changes in the tumor volume in xenografted SCID mice (n = 5/group) after injection of MDA-MB231 cells (1 × 107) were detected. Results are the means and standard deviations (*p < 0.05, Student’s t-test) (middle). Quantification of Ki67 positive cells after IHC analysis was performed (bottom) using image J software (NIH). *p < 0.05 (ANOVA). c Tumor growth measurement by treatment of oraparib in xenografted SCID mice (n = 5/group) at 28th day of MDA-MB-231 cells injection. *p < 0.05 vs. corresponding olaparib untreated control group and #p < 0.05 vs. olaparib untreated sh-control group (ANOVA). d IHC for BRCA1 and CTSS using human mammary cancer tissue microarray (n = 70), was performed using fluorescence-conjugated antibodies (CTSS: red; BRCA1: green). Photographs of four representative cancer tissues are presented. Quantification of BRCA1 positive and CTSS positive areas in each slide were analyzed using GraphPad Prism software 5.0
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