Regulating BRCA1 protein stability by cathepsin S-mediated ubiquitin degradation

Seoyoung Kim,Yun-Sil Lee,Hae June Lee,Hang-Rhan Seo,Hee Jin

doi:10.1038/s41418-018-0153-0

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.

Highlights

BRCA1, a tumor suppressor, participates in DNA double-strand break (DSB) repair, S and G2/M cell cycle checkpoints after damage, control of centrosome number, maintenance of heterochromatin, and transcriptional regulation of several genes [1,2,3]
Cathepsin S (CTSS) activity increased with 60 min of ionizing radiation (IR) (Fig. 1a), and IR resulted in dose dependent cleavage of inactive CTSS precursor (36 kDa) into its activated form (24 kDa), which is capable of degrading a range of macromolecules in MCF7 cells (Fig. 1b, c)
Immunofluorescence data indicated that V5-CTSS positive cells showed low expression of BRCA1 foci after IR than non-transfected negative cells (Fig. 1f). siRNA silencing of other cathepsins such as cathepsin L (CTSL) or cathepsin B (CTSB) did not result in inhibition of BRCA1 expression, and a pan inhibitor of cathepsin that can inhibit all cysteine cathepsins (E-64) did not fully restore BRCA1 expression when used at the same concentration as the CTSS specific inhibitor, VBY (Supplementary Figure S1C), suggesting that CTSS was activated by DNA damage response and inhibited BRCA1 expression

Summary

Introduction

BRCA1 (breast cancer susceptibility gene 1), a tumor suppressor, participates in DNA double-strand break (DSB) repair, S and G2/M cell cycle checkpoints after damage, control of centrosome number, maintenance of heterochromatin, and transcriptional regulation of several genes [1,2,3]. E Western blotting of cytosolic and nuclear fractions from MCF7 cells was performed. 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. Quantification of BRCA1 staining was performed by dividing into V5 positive and negative MCF7 cells. *p < 0.05 (Student’s t-test) regulates the stability of BRCA1 is the protein BARD1, which associates with BRCA1 to form a RING heterodimer that is essential for BRCA1 stability, nuclear localization, and E3 ligase activity [10] Another mechanism of BRCA1 degradation is HERC2-mediated BRCA1 ubiquitination. HERC2 interacts with the RING domain of BRCA1 and regulates BRCA1 stability in opposition to BARD1 [11]. The RING domain of BRCA1 mediates the interaction between BRCA1 and FBXO44. Over-expression of SCFFBXO44 reduces the BRCA1 protein level [12]

Methods

Results

Conclusion