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BRCA1 deficiency in triple-negative breast cancer: Protein stability as a basis for therapy

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Biomedicine & Pharmacotherapy 158 (2023) 114090

Available online 6 December 2022

0753-3322/© 2022 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Review

BRCA1 deficiency in triple-negative breast cancer: Protein stability as a basis for therapy

Eun Choi

a,1

, Gil-im Mun

a,1

, Joohyun Lee

a

, Hanhee Lee

a

, Jaeho Cho

b

, Yun-Sil Lee

a,*

aGraduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic of Korea

bDepartment of Radiation Oncology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea

A R T I C L E I N F O Keywords:

BRCA1 deficiency Triple-negative breast cancer Proteasomal degradation Chemotherapy resistance Multifunction

A B S T R A C T

Mutations in breast cancer-associated 1 (BRCA1) increase the lifetime risk of developing breast cancer by up to 51% over the risk of the general population. Many aspects of this multifunctional protein have been revealed, including its essential role in homologous recombination repair, E3 ubiquitin ligase activity, transcriptional regulation, and apoptosis. Although most studies have focused on BRCA1 deficiency due to mutations, only a minority of patients carry BRCA1 mutations. A recent study has suggested an expanded definition of BRCA1 deficiency with reduced BRCA1 levels, which accounts for almost half of all triple-negative breast cancer (TNBC) patients. Reduced BRCA1 levels can result from epigenetic modifications or increased proteasomal degradation.

In this review, we discuss how this knowledge of BRCA1 function and regulation of BRCA1 protein stability can help overcome the challenges encountered in the clinic and advance current treatment strategies for BRCA1- related breast cancer patients, especially focusing on TNBC.

1. Introduction

Breast cancer susceptibility gene 1 (BRCA1) is a well-known tumor suppressor gene, often mutated in familial breast and ovarian cancers [1, 2]. BRCA1 plays key roles in numerous cellular pathways involved in the maintenance of genomic stability, including DNA damage repair, DNA damage-induced cell-cycle checkpoint activation, transcriptional regu- lation, protein ubiquitination, chromatin remodeling, and apoptosis [3].

It is estimated that 20–25% of hereditary breast cancers and 5–10%

of all breast cancers are due to BRCA1 mutations [4]. Although BRCA1 mutations are rare (<5%) in sporadic tumors, high-grade breast cancers often demonstrate loss of heterozygosity (LOH) of BRCA1 [5]. Moreover, the frequency of BRCA1 mutation is higher (10–15%) in triple-negative breast cancer (TNBC). Over 75% of female breast cancer patients with BRCA1 mutation have a TNBC phenotype [6]. Promoter methylation (~30% of sporadic breast cancers and 15–57% of TNBC), somatic mu- tations, and gene deletion have been named as alternative mechanisms disrupting BRCA1 function and contribute to BRCA1-defective geno- types, displaying a biological and clinical phenotype similar to that of tumors harboring BRCA1 mutations. BRCA1-deficient breast cancers are generally high-grade and have poor prognoses [7]. In addition, 48–66%

of BRCA1 mutation carriers develop TNBC, rates that are much higher than that of non-carriers (~20%) [8–10]. The tight association between BRCA1 deficiency, basal-like phenotype, and TNBC calls into question whether loss of BRCA1 function plays any role in the development of sporadic TNBC, and whether such an association can be exploited therapeutically for the development of novel chemotherapeutics and biological agents.

Although BRCA1 deficiency due to germline or somatic mutations and hypermethylation of the BRCA1 promoter are relatively well defined in breast cancers, the regulation of BRCA1 protein stability in breast cancer is poorly described. Whereas BRCA1 mutations greatly increase the lifetime risk to develop breast cancer and affect chemo- therapy efficiency, dysregulation and altered expression of BRCA1 protein have been observed in sporadic forms of breast cancer, espe- cially in TNBC [11].

At this point, it would be meaningful to comprehensively summarize the current state of BRCA1 in terms of its structure and functions. More importantly, in this review, we explained BRCA1 deficiency accompa- nied by abnormal BRCA1 functions based on proteosomal degradation, and not with respect to BRCA1 genetic mutation, which has been the main focus of interest in BRCA1 research to date. In addition, BRCA1-

* Corresponding author.

E-mail address: yslee0425@ewha.ac.kr (Y.-S. Lee).

1 Equally contributed

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

https://doi.org/10.1016/j.biopha.2022.114090

Received 24 October 2022; Received in revised form 24 November 2022; Accepted 2 December 2022

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dependent apoptosis is mostly induced by BRCA1 accumulation in the cytoplasm. Therefore, increased cytoplasmic BRCA1 protein stability can be therapeutically useful for sensitizing TNBCs to chemo- and radiotherapy. Addressing these aspects would help the development of novel, BRCA1-mediated therapy in the future.

2. Structure of BRCA1

The BRCA1 gene consists of 24 exons with 1863 residues comprising two functional domains; an N-terminal really interesting new gene (RING) domain and tandem BRCA1 C-terminal (BRCT) domains [12].

The RING domain contains a RING finger motif and two flanking alpha helices. The RING finger motif contains a small, three-stranded, anti- parallel β-sheet and a central helix. Site I consists of four cysteine resi- dues and Site II of three cysteine residues and one histidine residue.

BARD1, one of the proteins that interacts through the N-terminal region of BRCA1, also has an N-terminal RING finger motif (residues 26–119).

Both BRCA1 and BARD1 require N-terminal RING finger regions for the formation of the heterocomplex. The N-terminal α-helix of BRCA1 and the C-terminal α-helix of BARD1 align in an antiparallel manner, while the C-terminal α-helix of BRCA1 is antiparallel to the N-terminal α-helix of BARD1 [13]. The RING finger is a module that interacts with E2s and enhances E3-ubiqutin (Ub) ligase activity of BRCA1 [14,15]. The for- mation of BRCA1-BARD1 heterodimer dramatically increases the Ub ligase activity of BRCA1 [16].

The BRCT domain is involved in interactions between BRCA1 and phosphoproteins, which are mostly phosphorylated by ATM and ATR kinases [17]. The BRCT domain is found as an isolated, individual domain, as a single or multiple tandem BRCT repeats, or even in com- plexes with other functional domains [18]. The BRCA1 BRCT domain recognizes the specific sequence of pSer-X-X-Phe in its phosphorylated binding partners, including BACH1, CtIP, and CCDC98/Abraxas [17,19, 20].

The central region of the BRCA1 protein, which covers over 60% of the BRCA1 sequence, is primarily encoded by a single, lengthy exon, exon 11 [21]. Exon 11 contains two nuclear localization signals (NLS) at

amino acids 503–508 and 606–615, which mediate BRCA1 translocation to the nucleus [22]. A nuclear export signal (NES) at amino acids 81–99 is required for nucleus-to-cytoplasm shuttling [23]. BRCA1 also contains a serine cluster domain (SCD) encompassing residues 1280–1524. This region has a high number of presumed phosphorylation sites, which are phosphorylated by ATM/ATR kinases. Phosphorylation of BRCA1 pro- motes the recruitment of BRCA1 onto sites of double-strand break (DSB) upon DNA damage [24]. The scaffold of the central region of BRCA1 has been predicted using the AlphaFold structure prediction tool but with very low model confidence [25,26]. The central region of BRCA1 in- teracts with various proteins and consists of a number of DNA damage-induced phosphorylation sites that are involved in the forma- tion of various BRCA1 protein complexes (Fig. 1).

3. BRCA1 functions

3.1. DNA repair and genomic stability

BRCA1 plays a role in several cellular processes, including the regulation of transcription, apoptosis, Ub ligation, transcription-coupled repair, and remodeling of chromatin. However, the well-known, tumor- suppressive function of BRCA1 is related to its role in promoting genomic stability. The hyperphosphorylation of BRCA1 leads to its rapid relocation to the sites of replication, with the recruitment of multiple, distinct protein complexes that can recognize and repair damaged DNA and activate cell-cycle checkpoints [27–30]. Various serine residues (S988, S1189, S1387, S1423, S1457, S1524, and S1542) are phosphor- ylated by Chk1, Chk2, ATR, or ATM [29, 31–35].

BRCA1 influences the path of cell to enter non-homologous end joining (NHEJ) or homologous recombination (HR) pathways to repair damaged DNA. Various studies have confirmed the direct role of BRCA1 in the HR pathway, as BRCA1-deficient cells show severely impaired, HR-mediated DSB repair [36,37]. The formation of the BRCA1-BARD1 complex is critical to the role of BRCA1 in DSB repair by HR. During the repair process, DSB ends are resected to produce 3single-stranded DNA tails [38]. These DNA tails are coated with replication protein A

Fig. 1. Structure of BRCA1. (A)Primary structures of BRCA1 and BARD1 both consist of a RING domain and a BRCT domain. BRCA1 and BARD1 form a heterodimer through their RING domains. The BRCA1 subunit is in purple, and the BARD1 subunit is in grey. The sites of I and II Zn2+atoms (green) are depicted (PDB ID: 1JM7) within each subunit. The crystal structure of the tandem BRCT repeats are in cyan (PDB ID: 1L0B). SCD in red encompasses the region of serine residues phos- phorylated by ATM/ATR kinases upon DNA damage.(B)Hypothetical three-dimensional structure of BRCA1, an intrinsically disordered protein, is predicted using the AlphaFold structure prediction tool and modified with PyMOL.

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(RPA), which is subsequently displaced by the recombinase protein RAD51, an essential protein in HR repair, promoting the downstream recovery pathway [39]. Then, DNA synthesis and the resolution of DNA intermediates occur to complete the repair [40]. BRCA1-BARD1 en- hances DNA invasion in HR by interacting directly with RAD51. In particular, BRCA1-BARD1 enables the capture of homologous duplex DNA by the RAD51–ssDNA nucleoprotein complex and assembles the synaptic complex, forming the displacement loop (D-loop) [39,41].

Also, BRCA1-BARD1 helps regulate cell-cycle checkpoints by antago- nizing 53BP1 during DNA end resection and promoting the activity of the resection nuclease MRN/CtIP [42,43] (Fig. 2).

3.2. Cell-cycle checkpoints

BRCA1 activates DNA damage checkpoints that occur in the G1/S,

intra-S, and G2/M phases when DNA is damaged, thereby contributing to cell survival. BRCA1 interacts with different proteins to activate each phases. Xu et al. emphasized the crucial role of BRCA1 in S phase checkpoint activity by demonstrating the impaired activity of S-phase checkpoints during DNA damage in BRCA1-deficient cells and the restoration of activity to the normal level by introducing the functional BRCA1 gene. BRCA1 participates in the S-phase checkpoint through the regulation of Chk1 kinase activity. Furthermore, S-phase checkpoint activation is associated with the phosphorylation of serine 1387 of BRCA1 by ATM, suggesting a possible involvement of phosphorylated BRCA1 in recruiting other regulatory components in the signaling cascade [28]. BRCA1 interacts with the MRN complex, which scans cells for DSBs and directly activates ATM [44,45]. The mutation of serine 1423 abolishes the ability of BRCA1 to induce G2-M arrest while retaining DNA repair function [28,46]. However, the ATM-dependent,

Fig. 2. Function of BRCA1. BRCA1 is involved in several functions, including 1) DNA repair, 2) cell-cycle checkpoint, 3) E3 ubiquitin ligase activity, and 4) apoptosis by interacting with various proteins. Circled numbers show six different specific pathways of BRCA1-mediated apoptosis: ① p53-dependent apoptosis pathway, ② Ras/MEKK4/JNK and Fas-dependent apoptosis pathways, ③ induction of GADD45-mediated MEKK4/JNK signaling pathway via nuclear export of BRCA1, ④ apoptotic calcium release via binding of BRCA1 to IP3Rs, ⑤ ERα-dependent apoptosis pathway via BRCA1-mediated ERα ubiquitination and degradation ⑥ cytosolic BRCA1-mediated proteasomal degradation of anti-apoptotic Bcl2.

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ionizing radiation (IR)-inducible S-phase checkpoint requires the phos- phorylation of serine 1387 of BRCA1, but not of serine 1423 [29]. Serine 988 mutation of BRCA1 can disrupt its function in HR but does not affect the S-phase checkpoint. Conversely, the serine 1423/1524 mutations are associated with normal HR, but lack G2-M phase checkpoint activity [46]. At the G1-S checkpoint, BRCA1 mediates the phosphorylation of p53 by ATM upon DNA damage, leading to the expression of p21, a CDK inhibitor. BRCA1-SIRT1 regulates the S-phase checkpoint by forming a reciprocal loop [47]. Furthermore, BRCA1-BACH1, another complex involved in S phase, is required for stalling replication forks due to DSB or DNA lesions [48,49]. The BRCA1-Abraxas-RAP80 complex activates the G2-M phase cell cycle checkpoint, triggering the phosphorylation of Chk1 [50]. Additionally, during the G2 phase, CtIP-BRCA1 contributes to the activation of the G2-M transition phase checkpoint and the phosphorylation of Chk1 upon DNA damage [51,52]. During mitosis, the Chk2-BRCA1 signaling cascade is activated downstream of DNA-PKcs and influences mitotic microtubule assembly [53] (Fig. 2).

3.3. Apoptosis

Many studies have demonstrated that BRCA1 plays a crucial role in inducing apoptosis through various mechanisms. Here, we emphasize BRCA1s role in inducing apoptosis to present evidence for restoring BRCA1 function in current treatment strategies for BRCA1-related can- cer patients. BRCA1 is a nuclear-cytoplasmic shuttling protein, while BARD1 plays a role in transporting BRCA1 into the nucleus. BRCA1- dependent apoptosis results when the BRCA1-BARD1 complex is dis- rupted and BRCA1 accumulates in the cytoplasm [54].

Two NLSs, in exon 11 of BRCA1, mediate the nuclear translocation of BRCA1 [55]. The translocation of alternatively spliced variants of BRCA1 with spliced-out exon 11 demonstrated an alternative pathway for the nuclear translocation of BRCA1 via its binding partner BARD1 [56]. Furthermore, the N-terminus of BRCA1 also has two NESs that promote the export of BRCA1 from the nucleus, mediated by chromo- some region maintenance protein 1 [57–59]. BARD1, directly masking the NES signal of BRCA1, exlpoits its own NLS to facilitate the nuclear localization of BRCA1. Moreover, BRCA1-binding protein 2 (BRAP2) binds to the BRCA1 NLSs to mediate cytoplasmic retention by disrupting the interaction with nuclear import receptor importin-α [60,61]. In the cytoplasm, BRCA1 regulates mitotic cell division, mitochondrial genome repair, cytoskeletal rearrangement, and apoptosis [62–64].

Therefore, the translocation of BRCA1 between cellular compartments is crucial for its function.

BRCA1-induced apoptosis, independent of p53, is triggered by BRCA1 nuclear export and proceeds through Ras-MEKK4-JNK and Fas- dependent signaling pathways that can activate caspase 8 [65].

Conversely, BARD1 reduces BRCA1-mediated apoptosis via nuclear sequestration. Upon DNA damage, BRCA1 exports to the cytoplasm in a p53-dependent manner. As more than 50% of solid tumors harbor p53 mutations, it is likely that genetically wild-type BRCA1 can function abnormally due to compromised nuclear-cytoplasmic shuttling in spo- radic breast cancer patients with dysfunctional p53. Furthermore, the BRCT region of BRCA1 is important for its interaction with p53, inter- fering the binding of BRCA1 and BARD1, leading to BRCA1 nuclear export [66].

BRCA1 undergoes various posttranslational modifications such as ubiquitination, phosphorylation, and sumoylation [67,68], which can affect its nuclear trafficking. The phosphorylation status of BRCA1 at serine 988 changes during cell-cycle progression, and the phosphory- lated form relocates to the perinuclear region of the cell in S-phase [69].

The cell cycle-related nuclear export of BRCA1 involves a calcium-dependent mechanism. BRCA1 is also involved in p53-dependent apoptosis by regulating the expression of p53-inducible gene 3 (PIG3), a downstream protein of p53. Clinical data suggest a significant association between PIG and BRCA1 expressions and the increased survival of breast cancer patients [70]. A correlation between

BRCA1 and the altered production of tumor necrosis factor-alpha (TNFα), an apoptotic inducer, has also been reported [71]. Natriuretic peptide receptor 3 inhibits cytoplasmic BRCA1 and TNFα and protects cardiomyocytes from apoptosis [72]. Estrogen (E)2/estrogen receptor (ER)α complex activates several pathways associated with cell-cycle progression and the prevention of apoptosis [73,74], while the E2/ERβ complex induces apoptosis of cells in many cases [74–76].

Furthermore, ERα induces anti-apoptotic signals, including the upre- gulation of Bcl2. BRCA1-BARD1 are required for ERα ubiquitination and degradation, and repression of either one results in ERα accumulation, illustrating a feedback loop between BRCA1-BARD1 and ERα since BRCA1-BARD1 formation is induced by ERα. Thus, BRCA1 has the po- tential to enhance apoptosis by modulating ERα [77]. BRCA1 binds to inositol 1,4,5-trisphosphate receptors (IP3Rs) to facilitate apoptotic calcium release. Calcium release mediated by IP3Rs regulates many signaling pathways, including those involved in apoptosis [78].

BRCA1 induces cell death by activating growth arrest and DNA damage 45 (GADD45) gene in a p53-independent manner. BRCA1 can either activate GADD45 through the interaction with Oct-1 and CAAT motifs of the GADD45 gene [79] or repress GADD45 through the inter- action with a Zinc finger protein, ZBRK1 [80]. Furthermore, the in- duction of BRCA1 triggers apoptosis through the activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway which is linked to GADD45 gene family members.

GADD45 binds and activates MTK1/MEKK4, which is upstream of the p38/JNK pathway. Therefore, BRCA1-induced apoptosis is also related to the p53-independent induction of GADD45 by BRCA1 and the acti- vation of JNK/SAPK [81].

In the cytoplasm, BRCA1 is detected at the centrosomes where it binds to γ − tubulin [83,84]. In addition, the presence of phosphory- lated BRCA1 in mitochondria and of BRCA1 complexed with Bcl2 in the endoplasmic reticulum has been confirmed [82–84]. The involvement of BRCA1 in apoptosis can also be explained by the relationship between BRCA1 and anti-apoptotic Bcl2 proteins. Nuclear BRCA1 facilitates the synthesis of Bcl2, while cytosolic BRCA1 promotes proteasomal degra- dation of Bcl2 [85]. This explains the important involvement of cytosolic BRCA1 in the induction of apoptosis upon DNA damage.

Interestingly, BRCA1 is found as a nuclear phosphoprotein in normal and tumor cells from tissues other than the breast and the ovary.

However, BRCA1 is predominantly cytoplasmic in breast and ovarian cancer cells [86]. The nuclear export of BRCA1 is particularly linked to its pro-apoptotic activity, which diminishes by the mutation of the N-terminal NES region and BARD1-mediated nuclear retention of BRCA1 [54]. Hence, it is hypothesized that the BRCA1-BARD1 hetero- dimer performs its cell survival function through its role in DNA repair, when localized to the nucleus, whereas BRCA1 and BARD1 individually induce apoptosis associated with their independent export to the cyto- plasm [54, 87–89]. Other studies have reported a correlation between cytoplasmic BRCA1 and the induction of the intrinsic caspase cleavage pathway following DNA damage [90,91]. The mechanism by which cytoplasmic BRCA1 induces cell death remains to be fully understood and is likely to involve several factors.

Reduction of the nuclear BRCA1 protein levels is found to have independently favorable effects on both the relapse-free (RF) and overall survival of patients. Cytoplasmic BRCA1 is not associated with classical, histological and morphological indicators except lymph node metas- tasis, and its relationship with the outcome of patient is reversed, pro- longing the patient’s RF survival. Thus, the prognostic significance of the BRCA1 protein differs according to its subcellular distribution.

Detection of BRCA1 protein in the nucleus is associated with poor prognosis, while cytoplasmic BRCA1 is linked to lower rates of recur- rence because of less lymph node metastasis [92]. Thus, modulating BRCA1 location can be therapeutically useful for sensitizing breast tu- mors to chemo- and radiotherapy. Translocating nuclear BRCA1 to the cytoplasm can be a useful strategy to sensitize p53-deficient, sporadic breast cancers to DNA damaging therapy (Fig. 2).

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3.4. Ubiquitination

The E3 Ub ligase activity of BRCA1-BARD1 presents a regulatory role in centrosome duplication and assembly of mitotic spindle pole [93–95].

BRCA1 has been reported to ubiquitinate a centrosomal protein, nucleophosmin (NPM1), which is important for centrosome duplication [96]. The BRCA1-BARD1 heterodimer mediates in vitro ubiquitination reactions and produces conjugates of BRCA1 primarily harboring lysine 6 (K6)-linked polyUb chains. K6-linked polyUb conjugates of BRCA1 are also generated in vivo by autoubiquitination [97,98]. The BRCA1-BARD1 heterodimer possesses intrinsic Ub E3 ligase activity that can autoubiquitinate BRCA1 [99–101] and ubiquitinate other substrates [102,103]. However, BRCA1 autoubiquitination, predominantly involving K6 of Ub for polyubiquitination, may not be directly respon- sible for its proteasome-sensitive degradation since the levels of these conjugates are not affected by proteasome inhibitors [97, 98, 103–105].

The BRCA1-BARD1 complex induces deposition of Ub signals on various substrate proteins, thereby acting as a RING type E3 Ub ligase.

Various types of Ub signals conjugated substrate proteins by BRCA1- BARD1 are involved in aspects of its function in DSB repair, cell-cycle regulation, and transcriptional regulation. A variety of BRCA1-BARD1 binding partners have been identified, any of which could also be sub- strates of BRCA1-BARD. However, only a subset of BRCA1-BARD1- binding proteins has been clearly demonstrated to be ubiquitinated and an even smaller subset confirmed to be BRCA1-BARD1-dependent substrates. The substrates of BRCA1-BARD1 include Aurora B, Cdc25c, claspin, CtIP, cyclin B, ERα, H2A, LARP7, macroH2A1, NF2, nucleo- phosmin, Oct1, p50, progesterone receptor, RPB1, BPB8, TFIIE, topo- isomerase IIa, and γ − tubulin [106] (Fig. 2).

3.5. Other functions

BRCA1 is involved in various mechanisms of transcription regulation [109]. BRCA1 can interact with the RNA polymerase II holoenzyme through RNA helicase A [107] and with the negative elongation factor complex through COBRA1 [108]. BRCA1 also interacts with diverse transcription factors, including p53 [109], c-MYC [110], ER [111], and GATA3 [112]. BRCA1 mediates chromatin decondensation through in- teractions with SWI/SNF remodeling complexes [113–115] and the histone acetyltransferases P300 and CBP [116]. Conversely, the BRCA1-BARD1 heterodimer is associated with chromatin condensation through interactions with the DNA methyltransferase DNMT3B and heterochromatin protein 1 [117,118]. BRCA1 is also linked to the regulation of chromatin structure. For instance, the dissociation of BRCA1 from chromatin driven by oncogenic RAS is known to promote senescence-associated heterochromatin foci (SAHF) formation during senescence [119]. BRCA1 causes large-scale chromatin decondensation by interacting with BRG1, which is the catalytic subunit of the SWI/SNF chromatin-remodeling complex and is known to regulate heterochro- matin structure [120].

4. BRCA1 deficiency; the expansion of definition

Studies of knockout mice have demonstrated that null mutations of BRCA1 lead to early embryonic lethality [121]. Germline BRCA1 mu- tations usually arise within one allele and the other wild-type allele of BRCA1 is sufficient to carry on most BRCA1 functions. However, the corresponding wild-type allele is further mutated or almost always lost in tumors that arise in BRCA1 mutant carriers, causing BRCA1 defi- ciency [122]. BRCA1 deficiency is a double-edged sword—although genome instability dysregulates critical tumor suppressors and onco- genic factors to promote tumorigenesis, too much DNA damage can initiate a lethal block by promoting apoptosis to hamper tumor forma- tion [123]. Therefore, the prognosis of BRCA1 pathogenic variant breast cancer is a subject of much debate. Some studies have indicated that BRCA1 pathogenic variants are linked to adverse prognoses [124–126],

whereas the predominant view is that the prognoses of BRCA1 patho- genic variant and sporadic breast cancers do not differ [127–129].

To date, more than 1600 mutations have been identified in the BRCA1 gene. The most frequent BRCA1 mutations are located within the BRCT and RING domains, as well as in exons 11–13, regions that are necessary for BRCA1 function and serving as binding sites of various BRCA1-interacting proteins, such as c-Myc, RAD50, pRb, RAD51, BRCA2, and PALB2 [21,130]. The most common BRCA1 mutations identified are 185delAG and 5382insC, frameshift mutations found in the RING and BRCT domains, respectively. [131,132]. Other common germline mutations of BRCA1, 3819del5 and 4153delA, were found on exon 11 [133].

The high prevalence of LOH of BRCA1 in tumors arisen in mutation carriers support the hypothesis of the classical tumor suppressor func- tion of BRCA1 [134]. As somatic mutations are rarely found [135,136], somatic loss of the wild-type allele has been suggested to be the common mechanism of BRCA1 inactivation, in addition to the germline mutation [134,137]. Several studies have revealed frequencies of LOH of BRCA1 ranging from 29.4% to 52% and provided evidence that haplo-insufficiency may explain the mechanism that contributes to the loss of the tumor suppressor function of BRCA1 in sporadic breast cancer [138–142].

TNBC is an aggressive subtype of breast cancer, defined by the absence of ER, progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. TNBC accounts for an estimated 10–15% of all breast cancers and is often associated with advanced disease stage and higher-grade tumors at diagnosis and an increased recurrence risk and poor 5-year survival rates compared to other breast cancers [7]. Inherited BRCA1 mutations predispose carriers to early-onset tumorigenesis and an up to 87% cumulative lifetime risk of developing breast cancer and/or ovarian cancer [143]. Germline genetic testing of TNBC patients using hereditary cancer gene panels is currently a common practice since 48–66% of BRCA1 mutation carriers develop TNBC, a rate that is much higher than that of non-carriers (~20%) [8–10]. However, even though BRCA1 mutation carriers are exposed to a higher risk of developing TNBC, only 10–15% of TNBC patients carry a BRCA1 mutation, which, though very high, indicates that over 85% of TNBC patients have at least one functional BRCA1 allele. Approximately 40–70% of TNBC tumors have been reported to gain a presumed HR deficiency phenotype, exceeding the prevalence of cases with germline or somatic BRCA1-inactivating variants [144]. This suggests that other mechanisms and/or genes may confer a similar phenotype, assuming that other regulatory processes may be involved in disrupting BRCA1 function.

The classical definition of BRCA1 deficiency has only taken mutation into account. However, comparing the actual incidence rate of BRCA1 mutation and the incidence rate of HR deficiency phenotype found in TNBC highlights the importance of understanding the expanded defi- nition of BRCA1 deficiency that results from the reduction of functional BRCA1. Low levels of BRCA1 expression are observed in an estimated 30% of sporadic breast cancers, perhaps due to hypermethylation of the promoter or other transcriptional regulatory mechanisms. Methylation of the BRCA1 promoter has been reported in many sporadic breast cancers. However, the involvement of BRCA1 has not been clearly elucidated in non-hereditary breast cancers, which account for 90% of all cases [145,146].

Hypermethylation of the BRCA1 promoter has been detected in 11–31% of all breast cancer cases. BRCA1 methylation was not detected in normal tissues or cell lines but has been observed exclusively in ma- lignant breast and ovarian tissues. Even though BRCA1 hyper- methylation varies in accordance with histological subtypes, it is commonly found in mucinous and medullary subtypes [145]. Interest- ingly, these histological subtypes are also frequently observed in inherited BRCA1 mutation carriers [147]. These cancers represent a specific phenotype, such as loss of ER and PR positivity, and are often accompanied by mutated p53. Hypermethylation of BRCA1 promoter

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has been reported in 16–57% of TNBCs across studies, superseding the prevalence of germline BRCA1 mutation, albeit with contrary reports of any association with prognosis [144]. However, some tumors with a decrease in BRCA1 expression showed no evidence of hypermethylation [148]. Therefore, a mechanism other than methylation may contribute to the decrease in BRCA1 levels detected in the majority of sporadic invasive breast cancer cases—the proteasomal degradation of BRCA1 (Fig. 3).

5. BRCA1 degradation

BRCA1 levels fluctuate in a cell cycle-dependent manner [97].

BRCA1 is phosphorylated and accumulates in the nucleus when cells enter S-phase [69,149,150]. BRCA1 remains well expressed during mitosis [97,149,151] and then, undergoes ubiquitination and proteasome-dependent degradation as cells enter the G1 phase [97, 149]. As cells progress from mitosis to the following G1 phase, ubiq- uitinated conjugates of endogenous BRCA1 polypeptides start to appear.

As these conjugates accumulate in cells upon the treatment with specific proteasome inhibitors, it is likely that proteolysis of BRCA1 is increased at the M-G1 transition and probably results in the downregulation of BRCA1 expression in early G1. This indicates that BRCA1 levels are regulated partly by the Ub/proteasome pathway and that turnover of

BRCA1 polypeptides is accelerated during the S phase [97].

The proteasome-sensitive Ub conjugates of BRCA1 are distinct from the autoubiquitination products of BRCA1, and their formation is probably catalyzed by other cellular E3 ligases [97]. Interestingly, the coexpression of BARD1 results in the formation of a stable heterodimer and inhibits the formation of these conjugates, indicating that BARD1 serves to stabilize BRCA1 levels, partly by reducing proteasome-sensitive ubiquitination of BRCA1 polypeptides. Moreover, the overexpression of BRCA1 can lead to an increase in BARD1 protein levels, which, in turn, can inhibit the ubiquitination of BRCA1 [15,152].

Throughout the G1 and S phases, steady-state levels of BARD1 fluctuate with cell-cycle progression in parallel with the levels of BRCA1. How- ever, BRCA1 is the more tightly regulated subunit of the BRCA1-BARD1 heterodimer because the half-life of BARD1 seems to be longer than that of BRCA1 [97].

BRCA1 is degraded by various Ub-proteasome-mediated pathways, including HERC2, HUWE1, FBXO44, and Parkin [103, 152–154].

HERC2, an E3 ligase, targets BRCA1 for degradation during the S phase [156]. Studies have shown that the BRCA1 level peaks in the late S and G2-M phases [97, 153, 155–158]. BRCA1 is mostly ubiquitinated and degraded during the S phase, and BRCA1 stabilization is accompanied by reduced ubiquitination during the G2-M phase [97,153]. HERC2 contributes to the ubiquitination and degradation of BRCA1, and BARD1

Fig. 3. Overview of BRCA1 deficiency in TNBC. The incidences of BRCA1 mutation and hypermethylation causing BRCA1 deficiency in TNBC patients are described.

The most common mutations of BRCA1 are depicted. Primary BRCA1 deficiency is derived by the loss of one wild-type allele and the concomitant mutation of the other allele. Additional causes of BRCA1 deficiency harboring wild-type BRCA1 are 1) epigenetic inactivation of the BRCA1 gene and 2) increased degradation of BRCA1 protein. (WT; Wild-type, Mt.; Mutant, LOH; Loss of Heterozygosity).

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is one of the regulators that affects HERC2-mediated proteolysis of BRCA1. The effects of HERC2 depletion on BRCA1 stability are remar- kable—BRCA1 levels are restored in the absence of BARD1. Conversely, BARD1 overexpression protected BRCA1 from ubiquitination by HERC2.

Furthermore, the binding partner for BRCA1 switches from HERC2 to BARD1 with an increase in BRCA1 steady-state levels during the period from the late S phase to mitosis. Thus, BRCA1 protein stability is tightly regulated by these two proteins, which is important for the cell’s pro- gression through the G2-M cell cycle checkpoint. BRCA1 ubiquitination decreases upon deletion of the BRCA1 N-terminal degron domain, indicating that ubiquitination depends on the degron domain. Although ubiquitination does not always lead to proteolysis, degron-dependent ubiquitination suggests that HERC2-mediated BRCA1 ubiquitination may be a signal for proteolysis [153].

HUWE1 also increases BRCA1 ubiquitination and degradation, thereby modulating BRCA1 activity. The binding site of HUWE1 within BRCA1 is located between amino acid residues 1 and 167 [152]. This N-terminal region of BRCA1 contains a degron domain that mediates the ubiquitination and instability of BRCA1 [104,152,153]. Wang et al.

observed that the immunoprecipitated BRCA1 with HUWE1 showed a high-molecular weight band shift, suggesting that HUWE1 may have contributed to a posttranslational modification of BRCA1. As HUWE1 is known to act as an E3 Ub ligase, this shift can occur due to ubiquiti- nation. Ubiquitination of BRCA1 may result from the interaction of the degron domain of BRCA1 and the NLS-containing acidic amino acid-rich region of HUWE1 (2000–2634 amino acids). Similar to HERC2, HUWE1 also targets BARD1-uncoupled BRCA1 for ubiquitination, but BARD1 is not a substrate for HUWE1 ubiquitination. Overall, HUWE1 targets BRCA1, but not BARD1, for polyubiquitination and degradation [152].

The Skp1-Cul1-F-box-protein44 (SCF(FBXO44)) complex is also associated with ubiquitination of the BRCA1 N-terminal degron domain.

The SCF complex, another E3 ligase, controls BRCA1 stability, suggest- ing that FBXO44 is an important F-box protein that mediates substrate recognition for BRCA1 ubiquitination. Unlike HERC2 or HUWE1, the SCF complex affects the stability of both BRCA1 and BARD1, and FBXO44 can recognize BRCA1 in a BARD1-independent manner since the abolishment of the interaction of BRCA1 with BARD1 does not affect the ubiquitination of BRCA1 by the SCF complex [103].

The Parkinson’s disease (PD)-associated protein kinase, PTEN- induced putative kinase1 (PINK1), and Ub E3 ligase Parkin, are com- ponents in a common signaling pathway regulating mitochondrial network homeostasis and mitophagy. Mitochondrial targeting reagents lead to mitochondrial depolarization along with the upregulation of PINK1 and proteasomal degradation of BRCA1. This BRCA1 degradation is controlled by PINK1, and BRCA1 downregulation upon mitochondrial damage causes DNA DSBs. BRCA1 degradation is facilitated by its direct interaction with an E3 ligase, Parkin. These observations indicate that mitochondrial damage is transmitted to the nucleus, causing BRCA1 degradation [154].

There are other regulators, other than E3 ligase, that affect the proteasomal degradation of BRCA1. Caspase-3, one of the most com- mon and important effectors of apoptotic processes, has been identified to mediate the cleavage of BRCA1, producing the C-terminus fragment of BRCA1 and reducing the levels of full-length BRCA1 during UV- induced apoptosis [159]. Another protease has been recently identi- fied to mediate the cleavage of BRCA1. Cathepsin S (CTSS), a lysosomal cysteine protease highly expressed in several cancer types, promotes ubiquitination and subsequent degradation of BRCA1 by cleaving its C-terminal BRCT domain. CTSS inhibition restores BRCA1 stability, thereby reducing tumorigenesis [160]. The C-terminus cleavage of BRCA1 by these proteases may enhance the proteasomal degradation of BRCA1 by producing N-terminal fragments of BRCA1.

The stability of the BRCA1-BARD1 complex is also important for the stability of BRCA1 itself and various regulators have been identified to affect this interaction. Sirtuin 2 (SIRT2), GUARDIN, and tumor- suppressor candidate 4 (TUSC4) enhance the stability of the BRCA1-

BARD1 heterodimer, whereas Ub-conjugating enzyme E2T (UBE2T) and transforming acidic coiled-coil 3 (TACC3) decrease the stability of the heterodimer.

SIRT2, a sirtuin family NAD+-dependent deacetylase, deacetylates BARD1 in the RING domain and enhances BRCA1-BARD1 hetero- dimerization [159]. GUARDIN, a p53-responsive, long, non-coding RNA, improves BRCA1 stability by promoting heterodimerization of BRCA1-BARD1 by acting as an RNA scaffold [161]. Furthermore, TUSC4 can directly interact with the E3 ligase HERC2, preventing BRCA1 degradation through the ubiquitination pathway. TUSC4 physically in- teracts with HERC2, a ligase of BRCA1, but not with BRCA1 [162].

Conversely, UBE2T interacts with the BRCA1-BARD1 complex and downregulates BRCA1 expression. BRCA1 was polyubiquitinated by incubation with the wild-type UBE2T protein, but not by C86A-UBE2T protein, an E2 activity-defective mutant [167]. High levels of TACC3 also result in reduced BRCA1 degradation. Thus, TACC3 inhibits the interaction between BRCA1 and BARD1 and allows the BARD1-uncoupled BRCA1 to be destabilized by Ub-mediated proteoso- mal degradation [163]. Even though the stability of the BRCA1-BARD1 heterodimer affects the stability of BRCA1 and its degradation, protea- somal degradation of BRCA1 can occur regardless of its interaction with BARD1 (Table 1).

6. Perspective of treatment strategy for overcoming resistance in BRCA1 deficiency

Defining groups of patients with BRCA1-deficient TNBC is important for the clinical management of patients since various new treatment strategies are investigated for BRCA1/2 mutation-related tumors. Tu- mors with defected BRCA1/2 proteins may be particularly sensitive to DNA damage-inducing agents, like platinum-based therapeutics, because the impairment of these proteins contribute to defective DNA repair by HR [164]. Furthermore, poly(ADP-ribose)-polymerase (PARP) inhibitors showed effective for the treatment of BRCA1/2-mutated tu- mors [165]. Although it is widely accepted that BRCA1 deficiency in TNBC leads to increased chemotherapy sensitivity, several studies have demonstrated that BRCA1 depletion increases resistance to DNA-damaging agents in animal models. Even though studies have shown conflicting results, BRCA1 complexity and its various biological functions imply that the final effect of BRCA1 on chemotherapy sensi- tivity will be greatly dependent on and specific to the tissue, its genetic background, as well as the therapeutic agent and its mechanism of ac- tion [166]. Here, we suggest several therapeutic strategies to enhance BRCA1-mediated apoptosis for the treatment of BRCA1-related TNBC patients with decreased BRCA1 levels.

We already mentioned that cytoplasmic BRCA1 is involved in the apoptotic pathway. BRCA1 stimulates p53-independent, GADD45- mediated apoptosis. Overexpression of BRCA1 may induce apoptosis through its nuclear export and excessive cytoplasmic BRCA1 accumu- lation [167–169]. Thus, suppressing the nuclear localization of BRCA1 may be a potential therapeutic strategy to sensitize tumor cells. Ectopic Table 1

Mediators of BRCA1 degradation.

E3 ligase/

protease Class Target region of

BRCA1 Involvement of

BARD1 References

HERC2 HECT N-terminal

degron Yes [153]

HUWE1 HECT N-terminal

degron Yes [152]

SCFFBXO44 RING N-terminal

degron No [103]

Parkin RING/

HECT N-terminal

degron unknown [154]

Caspase3 C-terminus

domain unknown [174]

CTSS BRCT domain unknown [160]

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expression of the RING domain fragment peptide allows nuclear export of BRCA1 to the cytoplasm [170]. The translocation of BRCA1 mediated by the RING domain fragment peptide reduces HR, sensitizing breast cancer cells to the EGFR inhibitor, erlotinib, and PARP1 inhibitors [171, 172]. This observation suggests that the RING domain fragment peptide can be a potential tool to deplete nuclear BRCA1 and improve the therapeutic response. Although there are various strategies to inhibit HR, it is important to specifically target the tumor and minimize normal tissue toxicity [173]. Our previous study suggested that CTSS interacts with the cytoplasmic BRCT domain of BRCA1 and induces proteasomal degradation of BRCA1. Treatment with CTSS inhibitors promotes apoptosis and radiosensitization in TNBC cells. Therefore, employing CTSS inhibitors for the inhibition of BRCA1 protein degradation in the cytoplasm may be a treatment strategy for the potentiation of BRCA1-induced apoptosis [160]. BRCA1-induced apoptosis is activated by BRCA1 nuclear export after the disruption of the BARD1-RCA1 interaction [168]. The BRCA1-BARD1 heterodimer activates a cell-survival signaling pathway when localized to the nucleus, partly due to its function in DNA repair. However, BRCA1 and BARD1 can individually facilitate apoptosis, consistent with their independent translocation to the cytoplasm [54, 87–89]. Therefore, the inhibition of the BARD1-BRCA1 interaction using small molecules may be a good strategy for the induction of BRCA1-mediated apoptosis in cancer cells.

7. Conclusions

It is evident that BRCA1 has an extensive role in maintaining genomic stability, thus preventing tumorigenesis. However, the inter- play among multiple cellular processes initiated by the loss of BRCA1 has made it challenging to distinguish between the causes and conse- quences of genomic instability in BRCA1-deficient cells and tumors and to determine which functions of BRCA1 are critical in its tumor sup- pressor function. These functions not only involve the established role of BRCA1 in HR but also its more recently described functions such as cell- cycle regulation, transcriptional regulation, ubiquitination, and apoptosis.

The decrease in BRCA1 expression, as observed in some sporadic cancers such as TNBC, is crucial in modulating chemosensitivity. The perception of BRCA1 as a positive modulator of sensitivity to DNA- damaging agents is consistent with its role in DNA damage repair.

Hence, cells with functional BRCA1 can be more effective in DSB repair by HR, implying that cancer therapy for BRCA1-defective patients could be improved by treatment with DNA-damaging agents. On the contrary, several studies showed that BRCA1 depletion increases resistance to DNA-damaging agents in animal models, and that the inhibition of cytoplasmic BRCA1 degradation is involved in apoptosis and sensitiza- tion to chemotherapy. Thus, enhancing BRCA1-mediated apoptosis may provide a novel therapeutic approach for the treatment of TNBC patients with BRCA1 deficiency.

The development of PARP1 inhibitors has given hope to patients with hereditary breast cancer with BRCA1 deficiency. However, tumors with acquired resistance require new therapeutic agents. To develop novel therapeutic agents, it is important to understand the mechanism of drug-resistant cancer development by harnessing functions other than DNA repair. Therefore, it is essential to clarify these mechanisms of BRCA1 at the molecular level to develop new approaches for diagnosis and treatment. Furthermore, in view of the fact that the intracellular functions of many proteins have not been elucidated fully, dysfunction of any protein in living organisms could cause disease. Thus, drug development based on the intracellular functions of BRCA1 could offer a new therapeutic approach for sporadic cancer, especially TNBCs, which lack suitable treatment targets.

CRediT authorship contribution statement

Eun Choi: Conceptualization, Methodology, Writing – original draft.

Gil-im Mun: Conceptualization, Methodology, Writing – original draft.

Joohyun Lee: Methodology, Writing – original Draft. Hanhee Lee:

Methodology, Writing – original Draft. Jaeho Cho: Visualization, Investigation. Yun-Sil Lee: Writing – review & editing, Supervision.

Declaration of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grants (NRF-2020R1A2C3013255 2018R1A5A2025286 and 2020M2D9A2093974), funded by the Korea government (Ministry of Science and ICT). The authors thank Medical Illustration & Design, part of the Medical Research Support Services of Yonsei University College of Medicine, for all artistic support related to this work.

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Although the nodal value for displacement coincides with the exact solution, the values between nodes are very inaccuracy in the case of using few elements due to using

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The index is calculated with the latest 5-year auction data of 400 selected Classic, Modern, and Contemporary Chinese painting artists from major auction houses..

1 John Owen, Justification by Faith Alone, in The Works of John Owen, ed. John Bolt, trans. Scott Clark, &#34;Do This and Live: Christ's Active Obedience as the

• Hormone Therapy With or Without Combination Chemotherapy in Treating Women Who Have Undergone Surgery for Node-Negative Breast Cancer (The TAILORx Trial)..

(Background) The standard triple therapy used as the first-line treatment for Helicobacter pylori infection are a combination of proton pump inhibitor(PPI),

As a result, in order to form a cooperation system for the regulation of shipment and production by structuring a network centering on Zakmokban

verify that USF2 interacts with BRCA1 in HeLa cells, and investigate changing interaction between.. BRCA1 and USF2 after treatment of ionizing radiation (IR), we