Deciphering the BRCA1 Tumor Suppressor Network

The BRCA1 tumor suppressor protein is a central constituent of several distinct macromolecular protein complexes that execute homology-directed DNA damage repair and cell cycle checkpoints. Recent years have borne witness to an exciting phase of discovery at the basic molecular level for how this network of DNA repair proteins acts to maintain genome stability and suppress cancer. The clinical dividends of this investment are now being realized with the approval of first-in-class BRCA-targeted therapies for ovarian cancer and identification of molecular events that determine responsiveness to these agents. Further delineation of the basic science underlying BRCA network function holds promise to maximally exploit genome instability for hereditary and sporadic cancer therapy.     

BRCA1和BRCA2与DNA损伤修复及转录调控

乳腺癌和卵巢癌敏感基因BRCA1和BRCA2与同源重组,DNA损伤修复,胚胎生长,转录调控及遍在蛋白化有关,其中,BRCA1和BRCA2在DNA损伤修复和转录调控中功能的确定,将有助于探讨和阐明两者的肿瘤抑制功能及其机理,作者将综述近年来有关BRCA1和BRCA2在DNA损伤修复和转录调控中功能研究的最新进展。     

Identification and functional characterization of a PP1-binding site in BRCA1

The phosphorylation state of the tumor suppressor protein BRCA1 is tightly associated with its functions including cell cycle control and DNA repair. Protein kinases involved in the DNA damage checkpoint control, such as ATM, ATR, and hCds1/Chk2, have been shown to phosphorylate and activate BRCA1 upon DNA damage. We reported previously that protein phosphatase 1alpha (PP1alpha) interacts with and dephosphorylates hCds1/Chk2-phosphorylated BRCA1. This study demonstrates the identification of a PP1-binding motif 898KVTF901 in BRCA1. Mutation or deletion of critical residues in this PP1-binding motif substantially reduces the interaction between BRCA1 and PP1alpha. PP1alpha can also dephosphorylate ATM and ATR phosphorylation sites in BRCA1 and may serve as a general regulator for BRCA1 phosphorylation. Unlike wild-type BRCA1, expression of the PP1 non-binding mutant BRCA1 protein in BRCA1-deficient cells failed to enhance survival after DNA damage. Taken together, these results suggest that interaction with PP1alpha is important for BRCA1 function.     

乳腺癌易感蛋白1在DNA损伤修复中的作用

人类乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)首先是在乳腺癌家族中发现的,是具有遗传倾向的乳腺癌和卵巢癌易感基因,其基因的突变与家族性乳腺癌及卵巢癌的发生有密切联系。BRCA1是一种抑癌基因,其基因产物可以参与维持基因组稳定性的多条细胞信号通路,例如DNA损伤诱导的细胞周期调控、DNA损伤修复、基因转录调节、细胞凋亡、泛素化等重要的细胞活动。本文就近几年来BRCA1在DNA损伤修复中的作用的研究进展作一综述,包括DNA损伤诱导的细胞周期检查点的激活和DNA损伤修复两方面。     

Targeting the lysosome by an aminomethylated Riccardin D triggers DNA damage through cathepsin B‐mediated degradation of BRCA1

RD‐N, an aminomethylated derivative of riccardin D, is a lysosomotropic agent that can trigger lysosomal membrane permeabilization followed by cathepsin B (CTSB)‐dependent apoptosis in prostate cancer (PCa) cells, but the underlying mechanisms remain unknown. Here we show that RD‐N treatment drives CTSB translocation from the lysosomes to the nucleus where it promotes DNA damage by suppression of the breast cancer 1 protein (BRCA1). Inhibition of CTSB activity with its specific inhibitors, or by CTSB‐targeting siRNA or CTSB with enzyme‐negative domain attenuated activation of BRCA1 and DNA damage induced by RD‐N. Conversely, CTSB overexpression resulted in inhibition of BRCA1 and sensitized PCa cells to RD‐N‐induced cell death. Furthermore, RD‐N‐induced cell death was exacerbated in BRCA1‐deficient cancer cells. We also demonstrated that CTSB/BRCA1‐dependent DNA damage was critical for RD‐N, but not for etoposide, reinforcing the importance of CTSB/BRCA1 in RD‐N‐mediated cell death. In addition, RD‐N synergistically increased cell sensitivity to cisplatin, and this effect was more evidenced in BRCA1‐deficient cancer cells. This study reveals a novel molecular mechanism that RD‐N promotes CTSB‐dependent DNA damage by the suppression of BRCA1 in PCa cells, leading to the identification of a potential compound that target lysosomes for cancer treatment.     

BARD1 regulates BRCA1 apoptotic function by a mechanism involving nuclear retention

BRCA1 is involved in maintaining genomic integrity and, as a regulator of the G2/M checkpoint, contributes to DNA repair and cell survival. The overexpression of BRCA1 elicits diverse cellular responses including apoptosis due to the stimulation of specific signaling pathways. BRCA1 is normally regulated by protein turnover, but is stabilized by BARD1 which can recruit BRCA1 to the nucleus to form a ubiquitin E3 ligase complex involved in DNA repair or cell survival. Here, we identify BARD1 as a regulator of BRCA1-dependent apoptosis. Using transfected MCF-7 breast cancer cells, we found that BRCA1-induced apoptosis was independent of p53 and was stimulated by BRCA1 nuclear export. Conversely, BARD1 reduced BRCA1-dependent apoptosis by a mechanism involving nuclear sequestration. Regulation of apoptosis by BARD1 was reduced by BRCA1 cancer mutations that disrupt Ub ligase function. Transfection of BRCA1 N-terminal peptides that disrupted the cellular BRCA1-BARD1 interaction caused a loss of nuclear BRCA1 that correlated with increased apoptosis in single cell assays, but did not alter localization or expression of endogenous BARD1. Reducing BARD1 levels by siRNA caused a small increase in apoptosis. Our findings identify a novel apoptosis inhibitory function of BARD1 and suggest that nuclear retention of BRCA1-BARD1 complexes contributes to both DNA repair and cell survival.     

Two redundant ubiquitin‐dependent pathways of BRCA1 localization to DNA damage sites

The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin‐dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80‐ and RING‐dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding‐deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80–BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.     

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3''-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.