53BP1: A key player of DNA damage response with critical functions in cancer

Accepted Manuscript Title: 53BP1: A key player of DNA damage response with critical functions in cancer Authors: Mohammad Mirza-Aghazadeh-Attari, Amir Mohammadzadeh, Bahman Yousefi, Ainaz Mihanfar, Ansar Karimian, Maryam Majidinia PII: DOI: Reference:

S1568-7864(18)30260-X https://doi.org/10.1016/j.dnarep.2018.11.008 DNAREP 2562

To appear in:

DNA Repair

Received date: Revised date: Accepted date:

13 October 2018 18 November 2018 19 November 2018

Please cite this article as: Mirza-Aghazadeh-Attari M, Mohammadzadeh A, Yousefi B, Mihanfar A, Karimian A, Majidinia M, 53BP1: A key player of DNA damage response with critical functions in cancer, DNA Repair (2018), https://doi.org/10.1016/j.dnarep.2018.11.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

53BP1: A key player of DNA damage response with critical functions in cancer Mohammad Mirza-Aghazadeh-Attaria,b, Amir Mohammadzadeha,b, Bahman Yousefic,d, Ainaz

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Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran;

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Mihanfare, Ansar Karimianf, Maryam Majidiniag,*

Aging Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran;

Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

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Department of Biochemistry, Faculty of Medicine, Urmia University of Medical Sciences,

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Urmia, Iran; f

of Medical Sciences, Babol, Iran; g

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Cellular and Molecular Biology Research Center, Health Research Institute, Babol University

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Solid Tumor Research Center, Urmia University of Medical Sciences, Urmia, Iran.

*Corresponding authors:

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Maryam Majidinia, Solid Tumor Research Center, Urmia University of Medical Sciences, Urmia, Iran. Tel: (+98)9033916051. Email: [email protected]

Abstract

Maintenance of genome integrity and stability is a critical responsibility of the DNA damage response (DDR) within cells, such that any disruption in this kinase-based signaling pathway leads to development of various disorders, particularly cancer. The tumor suppressor P53-binding

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protein 1 (53BP1), as one of the main mediators of DDR, plays a pivotal role in orchestrating the choice of double-strand break (DSB) repair pathway and contains interaction surfaces for numerous DSB-responsive proteins. It has been extensively demonstrated that aberrant expression of 53BP1 contributes to tumor occurrence and development. 53BP1 loss of function in tumor tissues is also related to tumor progression and poor prognosis in human malignancies. Due to

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undeniable importance of this protein in various aspects of cancer initiation/progression,

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angiogenesis, metastasis and development of drug resistance, as well as its targeting in the

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treatment of cancer, this review focused on explaining the structure and function of 53BP1 and its

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contribution to cancer.

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Keywords: 53BP1; DNA damage response; DNA repair; Double strand breaks; Cancer

1. Introduction The tumor suppressor P53-binding protein 1 (53BP1) is a member of Tudor-containing proteins (including JMJD2A, 53BP1, SGF29, Spindlin1, UHRF1, PHF1, PHF19 and SHH1) in ‘reading’

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unique methylation events on histones in order to facilitate DNA damage repair or regulate transcription (1). This protein is also one of the mediator/adaptor proteins of the DNA damage response (DDR), which includes the mediator of the DNA damage checkpoint protein 1 (MDC1), 53BP1, breast cancer-associated gene 1 (BRCA1), topoisomerase II-binding protein 1 (TOPBP1), Claspin and Pax transactivation domain-interacting protein (PTIP) (2). 53BP1 was first described

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as a binding partner of the p53, almost 25 years ago (3). 53BP1 is a key component of DDR and

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plays a pivotal role in orchestrating the choice of double-strand break (DSB) repair pathway; it

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contains interaction surfaces for numerous DSB-responsive proteins (4, 5). This protein has no

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characterized enzymatic activity and acts as a recruitment platform for other DDR proteins (2, 6).

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Access of 53BP1 to DSB is modulated by the Tudor-interacting repair regulator (TIRR) through

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masking the dimethylated lysine 20 of the histone H4 (H4K20me2) binding surface on 53BP1 (7). 53BP1 promotes non-homologous end-joining (NHEJ)-mediated DSB repair and prevents

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homologous recombination (HR) by counteracting the function of BRCA1 in the HR pathway (8).

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Interestingly, promotion of microhomology-mediated end-joining (MMEJ) by 53BP1 in G1-phase cells is observed in the presence of the functional BRCA1 (9). A defect in 53BP1 induces DNA

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damage checkpoint defects, impaired DNA repair and hypersensitivity to ionizing radiation (IR) (10, 11). Moreover, depletion of 53BP1 results in a cell cycle arrest in the G2/M phase and instability in human cells (12). 53BP1 is also essential for the adaptive immune system because of its role in NHEJ of distal DNA ends generated during long-range V(D)J recombination and class switch recombination (CSR), which are important for a functional adaptive immune response (13).

Various studies have shown the role of this protein in various cellular functions. For example, aberrant expression of 53BP1 was found to contribute to tumor occurrence and development. It was revealed that 53BP1 loss in tumor tissues was related with tumor progression and poor

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prognosis in breast cancer, and expression of 53BP1 was correlated with apoptosis in colorectal cancer (14) (15). Furthermore, it has been recently observed that 53BP1 is involved in regulating mitochondrial clearance from the cell via a type of autophagy termed mitophagy (16). In addition to the role of this molecule in the field of cancer, its function in the aging process can be highlighted by its association with lamin A/C (17). Regarding the structure and function of this protein, various

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studies have been published so far; however, the role of this molecule in various cancers has less

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been mentioned. Therefore, the present review focused on various roles of this protein in different

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aspects of cancer including initiation/progression, angiogenesis, metastasis and development of

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drug resistance.

The protein structure

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2.1.

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2. The structure, genome, and transcription of 53BP1

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53BP1 is a large protein with 1972 amino acids and over 200 kDa mass that is encoded by the TP53BP1 gene (18) (19). Important structural elements in 53BP1 include two repeats of BRCA1

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carboxy-terminal (BRCT), the tandem Tudor domains, a glycine/arginine-rich region (GAR) methylation stretch, two dynein 8 kD light chain (LC8) binding sites, and numerous

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phosphorylation sites including 32 PIK kinases and 41 cyclin-dependent (CDK) phosphorylation sites (called amino-terminal Ser/Thr-Gln (S/TQ) sites and Ser/Thr-Pro (S/TP) sites, respectively (20)), which are phosphorylated, at least in part, by the ataxia-telangiectasia mutated (ATM) kinase (18). 53BP1 was originally identified in a yeast two-hybrid screen as a protein that interacted with

the p53 DNA binding domain through its two C-terminal BRCT motifs (21). BRCT domains are 100–150 residue motifs found in a large number of proteins involved in the cellular responses to DNA damages (22, 23). GAR of 53BP1 is methylated by the protein arginine N-methyltransferase

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1 (PRMT1) (24, 25) and has been implicated in a possible DNA-binding function for 53BP1 (24). Some of S/TQ sites have been identified as phosphatidylinositol 3-kinase-related kinase (PIKK)dependent phosphorylation sites following ionizing radiation (IR) (26-28). Some of these sites have characterized roles in DDR signaling and protein interactions. For example, phosphorylation of 53BP1 at Ser25 is required for the interaction between 53BP1 and mediator PTIP, and abrogation

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of this interaction results in DNA-damage-sensitivity and reduced checkpoint kinase 2 (CHK2)

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phosphorylation (29). Additionally, phosphorylation of 53BP1 at Ser1219 has been reported to

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function in DNA damage signaling and G2/M-phase arrest following IR (28). 53BP1 exhibits

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strong functional similarity to MDC1 (30), BRCA1 (31) and their yeast orthologues (Rad9 in Saccharomyces cerevisiae and Crb2 in Schizosaccharomyces pombe) (22, 32).

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The gene structure

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2.2.

The TP53BP1 gene is on the chromosome 15 and its location is 15q15.3 with 32 exons (33).

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Expression of this gene in the brain is more than that of other tissues (34). Homologs of this gene are conserved in chimpanzees, Rhesus monkeys, dogs, cows, mice, rats, chickens, and frogs; a

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number of 239 organisms have orthologs with the human gene TP53BP1 (19, 35, 36).

Regulation and post-translational modification

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2.3.

53BP1 is continuously expressed in the cell nucleus (37) and accumulated in the nucleoplasm. 53BP1 has two subpopulations after DNA damage: the bulk 53BP1 in the nucleoplasm and 53BP1 bound at the damage site (38, 39). Since the bulk 53BP1 is not bound to chromatin epitopes at the damage site, it would be susceptible to ubiquitination and degradation. Bulk 53BP1 prevents the

RAP1-interacting factor 1 (RIF1) from binding to the DNA in the undamaged state. After IR, bulk 53BP1 is degraded, and RIF1 is recruited to damage sites by the bound 53BP1 to execute inhibition of end resection (40-42). RIF1 is one of a few identified proteins which requires 53BP1 for its

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recruitment to DSBs (43) and is involved in C-NHEJ (42). The protein level of 53BP1 at different stages of the cell division cycle is relatively stable with only minor oscillations (44, 45), suggesting that 53BP1 is being constantly transcribed and translated during the cell cycle. On the other hand, a number of studies have reported the post-translational regulation of 53BP1.

Three pathways regulate the cellular levels of 53BP1: an endosomal/lysosomal protease Cathepsin

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L (CTSL) that cleaves 53BP1 (46-48), UbcH7 that regulates the proteasome-dependent

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degradation of 53BP1 (49) and lamin A/C, the depletion of which reduces 53BP1 stability (50).

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Pozo et al. recently showed that UbcH7-dependent proteasomal degradation is the major pathway

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that controls the level of 53BP1 in the absence or presence of DNA damage in a wide range of cultured human cell lines (51). The mechanism by which lamin A/C protects 53BP1 is shielding it

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from the UbcH7-dependent degradation machinery (50, 51).

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53BP1 is a rare example of a protein whose post-translational modification-binding function can

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be switched on and off. Activity of 53BP1 is directly inhibited by 53BP1-binding protein TIRR (52, 53). X-ray crystal structures of TIRR and a designer protein bound to 53BP1 reveal a unique

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regulatory mechanism, in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. This 53BP1 inhibition is

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relieved by TIRR-interacting RNA molecules, providing the proof-of-principle of the RNAtriggered 53BP1 recruitment to DSBs (54). Furthermore, 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction between the two proteins is reduced after DNA damage. NuMA prevents 53BP1 accumulation at DNA breaks. Manipulating

NuMA expression alters the PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1(55). There are several mechanisms that limit the accumulation of 53BP1 at sites of DNA damage by

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preventing self-perpetuating activation of DNA damage checkpoints and excessive spreading of DNA repair factors to undamaged chromatin. Several de-ubiquitylating enzymes (DUBs) have been found to counteract DNA damage-dependent histone ubiquitylation and the assembly of 53BP1 at sites of DNA damage. These include BRCC36, USP3, POH1, and USP44 (56-59). Another DUB, called OTUB1, restricts histone ubiquitylation and 53BP1 loading, but in a non-

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catalytic manner that involves binding to UBC13 and hindering the ubiquitin ligase activity of

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RNF8 and RNF168 (60, 61). USP16 and USP28 have also been shown to modulate DDR and

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repair pathways. USP16 mediates histone deubiquitylation-dependent transcription silencing at

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DSB sites (62), and USP28 possibly regulates 53BP1 stability (59, 63). In addition, histone acetylation is thought to play a part in inhibiting 53BP1 chromatin binding. The acetyltransferase

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Tip60/Kat5 can acetylate histone H4 on lysine 16, which interferes with the binding of 53BP1 to

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the nearby H4K20Me2 (64, 65). Importantly, the H4K16 acetylation concomitantly increases the

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BRCA1 recruitment to DNA damage foci; moreover, the knockdown of Tip60/Kat5 or chemical inhibition of histone deacetylases (HDACs) can rescue the homology-directed repair (HDR) defect

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of BRCA1-deficient cells, similar to the absence of 53BP1 (59, 64, 66, 67). After detection of DSBs, 53BP1 rapidly accumulates on the chromatin surrounding the break site

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or at ionizing radiation-induced foci (IRIF) after IR-induced DNA damage (37, 59), because a cascade of protein modification and relocalization is triggered: this signaling cascade is initiated by the ATM-mediated phosphorylation of the histone 2A (H2A) variant H2AX (γH2AX), followed by the recruitment of MDC1 and activation of RNF8 (ring finger 8)-RNF168-dependent chromatin

ubiquitylation (68-70). Phosphorylation of H2AX (γH2AX) results in the recruitment of downstream factors, such as the E3 ubiquitin ligases RNF8 and RNF168, leading to the formation of K63-linked polyubiquitin chains on histones at DSBs (71, 72). This ubiquitination cascade

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regulated by RNF8 and RNF168 is responsible for the localization of repair mediators (73, 74), including BRCA1 and 53BP1, to DNA damage sites (73, 75). Although the initial recruitment of 53BP1 occurs independently of γH2AX (76). The stable association of 53BP1 with DSBs strongly relies on the RNF8-RNF168-mediated ubiquitylation cascade (73, 77), that is the activated downstream of γH2AX and MDC1 (78, 79). Yiheng et al. found that RNF8 and RNF168 not only

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mark histones at the break site to create a 53BP1 binding site, but these ubiquitin ligases also

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regulate the proteasome-mediated degradation of 53BP1 (80). Failure to degrade 53BP1 not bound

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to DSBs leads to mislocalization of a downstream factor RIF1, thereby impairing DSB repair (80,

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81). Interestingly, the C-terminal BRCT repeats of 53BP1, which mediate the interactions between p53 and EXPAND1, are dispensable for the focal recruitment of 53BP1 to damaged chromatin

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(82-84). The chromatin modulator EXPAND1 (also known as MUM1) is one of the two proteins

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that rely on 53BP1 for their accumulation at DSB sites, which interacts with the BRCT domains

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in 53BP1 in a phosphorylation-independent manner (85). The second protein is RIF1, which interacts (directly or indirectly) with ATM-phosphorylated residues in 53BP1 (86). Instead of the

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C-terminal BRCT repeats, the minimal focus-forming region of 53BP1 contains an oligomerization motif and tandem Tudor domains that bind to mono and dimethylated H4K20

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(H4K20me1 and H4K20me2, respectively) as well as an adjacent ubiquitylation-dependent recruitment (UDR) motif that binds to the RNF168-ubiquitylated H2AK15 (36, 87, 88). The domain structure of this minimal focus-forming region in 53BP1 reflects the need to integrate

multiple, independent histone marks that cooperate to recruit 53BP1, specifically to damaged chromatin (19).

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3. DNA damage response and 53BP1 DDR is a plethora of molecules active in a signaling cascade, where DNA damage is sensed, and molecules are translated to actions such as apoptosis, cell cycle arrest, senescence and most importantly, DNA repair via a specific transducer and mediator (89-91). DNA repair is classified to HR, mismatch repair, nucleotide excision repair, base excision repair and NHEJ (92, 93). It has

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been shown that alterations in the normal function of DDR can lead to defects in DNA repair or

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disruption of apoptosis or cellular arrest, which could be the starting point for neoplastic cell

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formation (91), or other human pathologies, aging and hereditary disorders (94, 95). 53BP1, is a

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molecule active as a DNA damage sensor and damage mediator, which has important roles in signaling of DDR transducers and mediators in the DSB arm, such as ATM, BRCA1 and p53 (96).

Interactions with molecules involved in DDR

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3.1.

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the cascade.

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Figure 1 further elicits the role of 53BP1 in DDR and its interactions with multiple molecules in

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3.1.1. Interactions with DDR sensors The Mre11/Rad50/Nbs1 (MRN) is the DNA damage sensor in the DSB arm of DDR, and has

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critical roles in activating downstream kinases and DDR transducers, namely ATM (97). A study by Lee et al. revealed that the interaction between the tandem breast cancer carboxy-terminal repeats of 53BP1 and the MRN complex increased the rate of ATM activation by phosphorylation and the subsequent downstream activation of molecules such as CHK2. Their study also showed that the interaction was facilitated by RAD50 from the complex, and led to the hyper

phosphorylation of Nbs1, and 53BP1 itself. Furthermore, their study indicated that the combined function of 53BP1, MRN complex and ATM was necessary for optimum DDR and DSB repair (98). The RPA complex is a DNA damage sensor in the single strand damage pathway of DDR. A

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study by Yoo et al. showed that there were important interactions between this complex and 53BP1. The researchers found that 53BP1 was involved in the phosphorylation of RPA2 following DNA damage. This was found by inducing DNA damage in U2OS osteosarcoma cells, in which 53BP1 was knockdown by siRNAs. These cells had an augmented apoptosis with camptothecin and showed increased sensitivity (99).

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3.1.2. Interactions with DDR transducers

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ATM is the main transducer of the DSB signal in the DDR cascade, and is activated by the presence

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of DSB. This activation is done in two manners, one is dependent on the activation of the MRN

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complex and the other is dependent on the change in the chromatins structure, which is mediated

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by 53BP1 (100). A study by Wilson et al. showed that 53BP1 mediates the ATM

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autophosphorylation in ATM S1981, a role which is unique among proteins with BRCT domains. 53BP1 is also able to induce T68 phosphorylation in CHK2, the downstream molecule in ATM

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signaling (31). Further, 53BP1 is phosphorylated by ATM in multiple residues, and by ATR in instances of UV-caused DNA damage (27). A study by Shibata et al. showed another relation

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between 53BP1 and the two transducers of DNA damage, the ATM and ATR. It was shown that

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cells needed the signaling of both ATM and ATR in order to maintain G2/M checkpoint arrest, and in high doses of exposure to radiation which triggered cell arrest, there was a need for the function of 53BP1 and MDC1. The authors concluded that lack of 53BP1 enhanced chromosome breakage in response to radiation (101). ATR is the equivalent of ATM in single strand DNA damage signaling, which exerts its role by affecting CHK1, a downstream transducer. Studies have

shown that 53BP1 mediates the interaction between CHK1 and ATR in instances of replication stress, which leads to the protection of replication forks. An absence of 53BP1 causes a defective ATR-Chk1-p53 signaling, leading to increased rates of apoptosis in ex vivo B cells (102).

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Interestingly, a study by Martinez et al. showed that a double lack of 53BP1 and TRF1, which protects telomeres from damage and fusion, caused an increased ATR function and CHK1 phosphorylation coupled with an increased HR (103). 3.1.3. Interactions with BRCA-1

Studies have shown that the effect of 53BP1 and BRCA-1 on each other has important outcomes

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regarding DDR signaling, namely DNA repair. A study by Xu et al. revealed that these two

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molecules governed the entrance into two distinct pathways for stalled replication restart. 53BP1

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promotes a fork cleavage-free pathway while BRCA-1 coupled with SLX-MUS promotes a break-

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induced replication (BIR) pathway (104). Further, these two molecules antagonize each other in

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DSB repair pathway selection. BRCA-1 promotes HR by co-localizing with H2AX to the site of

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damage and negatively regulating Mre11, a molecule which is part of the MRN complex and possesses nuclease activity. BRCA-1 also has profound roles in the transcriptional response to

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DNA damage, which further contribute to the HR progression (105). Further, BRCA-1 antagonizes BP531 by limiting its interaction to the chromatin in the S phase, thereby inhibiting 53BP1

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mediated repair (106). Regarding this, another study found that BRCA-1 was able to stop the

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translocation of RIF1 to breaks in the S2/G phase. This study also found that BRCA-1 needed its two most important domains, i.e. the RING and BRCT domains, to regulate the function of 53BP1 (107).

3.1.4. Interactions with p53 p53 is the centerpiece in DDR and activated downstream signaling cascades, which determines the final fate of cells undergoing DNA damage (108). Studies by Cuella-Martin et al. showed that p53

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had rigorous transcriptional activities, which were dependent on 53BP1 function. It was revealed that the transcription of molecules such as p21 and MDM2 was dependent on the function of p53, which, itself, was dependent on sufficient 53bp1 activation (109, 110). The same team also uncovered that the interaction between the two molecules was mediated by the BRCT and Nterminal domains of 53BP1, and also by a deubiquitylase called USP28 (109, 111, 112). Al Rashid

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et al. showed that the non-specific DNA binding domain of p53 in the C terminal of the molecule

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was responsible for these interactions (113). Interestingly, this function of 53BP1 is shown to be

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independent of the role of 53BP1 in DNA repair (114). Further evidence regarding the concomitant

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action of these two molecules is shown in a study by Morales et al. They found that animals with deficiencies in the function of both p53 and 53bp1 showed increased rates of T cell lymphomas

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and increased genomic instabilities in solid tumors compared to animals defective in only one of

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these molecules (115). Another study conducted by Ward et al. revealed similar results. Further,

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they suggested a 53-indipendent role for 53BP1 in antagonizing carcinogenic events (116). Another functional significance of 53BP1 in p53 action has been shown regarding cell cycle arrest.

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A study by Fong CS et al. showed that 53BP1 and USP28 had essential roles in mediating a p53 dependent cell cycle arrest in G1 in response to prolonged mitosis and loss of centrosome (117).

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Similar results were also obtained by Meitinger et al. (118) and Lambrus et al. (119). 3.1.5. Interactions with DNA repair As mentioned, DNA repair is one of the end points of DDR. 53BP1 is active in regulating DSBs damage, and its most important function is to promote NHEJ and suppress HR (120). In performing

this function, 53BP1 has close ties with BRCA-1, as mentioned previously. The initial step in initiation of repair is the recruitment of 53BP1 to the damage cite via interactions with MDC1, which is then sustained in the region by interactions with H4K20me2. Studies have also shown

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that RNF8 and RNF168 are necessary for recruitment (58, 87, 121). The main involvement of 53BP1 in DNA repair is the choice between NHEJ and HR. These two processes of DNA repair differ in the fact that HR is an error free process while NHEJ is not; however, NHEJ is shown to be a faster alternative to HR in DNA repair (122). After ATM phosphorylates 53BP1, a series of effector molecules such as RIF1 and PTIP prevent resection of DNA 5′ ends and promote NHEJ

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3.1.6. Interactions with apoptosis and autophagy

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(123). Further, 53BP1 itself inhibits end resection dependent on CtIP (59, 66).

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Apoptosis is defined as the programmed death of cells. DDR cascade has essential roles in the

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progression of apoptosis. Mutations in DDR can translate into functional variations in the process

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of apoptosis, which enable cells with compromised DNA content to avoid death and possibly

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acquire malignant characteristics (124). Evidence regarding the role of 53BP1 in apoptosis has been shown in C. elegans. A study by Ryu et al. analyzed the function of HSR-9, the homolog of

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53BP1, after exposure to ionizing radiation and found that HSR-9 promoted apoptosis and a form of the RAD-54 dependent NHEJ. This molecule has no independent role in cell cycle arrest (125).

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Hong et al. found that increased expression of 53BP1 in ovarian cancer cells increased the

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expression of molecules such as Bax, P21 and caspase 3 but decreased that of BCL-2 and AKT, having a net effect of promoting apoptosis (126). Another set of observations revealed that inhibiting the function of 53BP1 could increase apoptosis in cells undergoing DNA damage inducing agents. Yang et al. found that utilizing a glycogen synthesis kinase 3 (GSK3) β (which

phosphorylates and activates 53BP1) inhibitor called SB216763 would render glioblastoma cells more sensitive to apoptosis (127). Autophagy is defined as the clearance of unneeded cellular particles under conditions of stress.

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This function has multiple cross talks with DDR and its end points (128). A study by Youn et al. showed that 53BP1 had p53 independent roles in promoting mitophagy, a subtype of autophagy in H1299 and HeLa cells. They study found that cells with absent 53BP1 had mitochondria with abnormal shapes and mitochondrial aggregation coupled with an increased mitochondrial

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membrane potential (129).

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3.1.7. Interactions with cell cycle

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As mentioned, 53BP1 interacts with p53 and USP28 to contribute to cell cycle arrest. However,

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53BP1 also has other roles in controlling cell cycle. It was shown that 53BP1 was a target of cell cycle kinases, such as Polo-like kinase-1, which interacted with 53BP1 and led to a successful

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inactivation of the G(2)/M checkpoint (44). A study by Stiff et al. suggested that ATR enforced

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the G2/M arrest by the mediating effects of 53BP1, MDC1 and Nbs1 (130). Regarding the ATR pathway in DNA damage, TopBP1 is a mediator for the effects of 53BP1 in the G1, as shown by

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Cescutti et al. (131). A study by Kwak et al. demonstrated that the role of 53BP1 in upholding the

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mitotic checkpoint was dependent on the presence of the Jun activation domain-binding protein 1 (Jab1), which is a 53BP1 binding protein. They found in their study that the knock-down of Jab1

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resulted in reduced mitotic arrest in resistance to chemotherapy. It was hypostatized that this particular interaction could be a target of therapies aiming to sensitize cells to microtubuleinterfering anticancer drugs (132). In addition, a study by Sengupta et al. showed that 53BP1 had important functional interactions with the BLM helicase in upholding the S phase checkpoint. It

was shown that BLM was essential for 53BP1 and p53 interaction and that ATR/CHK1 was responsible for mediating the initial interaction between the BLM helicase and 53BP1 (133, 134).

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4. 53BP1 interaction with signaling pathways Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling: NF-κB signaling is one of the most important signaling pathways regarding human malignancies (135). It has been shown that NF-κB signaling induces cellular proliferation, angiogenesis, epithelialmesenchymal transition, metastasis and alterations in tumor microenvironment, which further

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promote cancer formation (136). DNA damage is one of the activating agents of NF-κB, which

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together with cell cycle proteins, initiates events that insure cell survival. This series of events is

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one of the reasons why tumors gain the ability to tolerate chemotherapy agents (137). The

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regulatory role of DDR on NF-κB signaling as well as the role of 53BP1 in this relation was shown in a study by Li et al. where 53BP1 activation inhibited NF-κB signaling, which led to decreased

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rates of tumor progression and metastasis. This regulatory effect (138) was imposed by miR-146a

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(139). Moreover, it has been proposed that NF-κB can have regulatory effects on DDR. A study

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showed that this signaling pathway could interact with ATM and BRCA-1 in promoting HR by promoting BRCA-1 stabilization and interaction with CtIP (140). Another study revealed that NF-

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κB mediated the expression of Ku70 and Ku80, the proteins involved in NHEJ (141). More evidence regarding the role of NF-κB in DNA repair was proposed by Kraft et al. who found that

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inhibition of NF-κB negatively affected both NHEJ and HR, and that this effect was cell specific and also different pattern of DSB repair was observed based on the cell type and content of other DDR molecules such as p53 (142).

AKT signaling: Another cellular signaling pathway with meaningful interactions with DDR and 53BP1 is the AKT signaling cascade (143, 144), which has established roles in human pathologies, specifically cancer (145, 146). A study by Liu et al. revealed that AKT mediated the

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phosphorylation of the X-ray repair cross complementing 4 (XRCC4)-like factor (XLF), leading to its disassociation from the XRCC4/DNA ligase IV complex. This resulted in the impairment of the functional goal of 53BP1and NHEJ. Further, it was shown that a mutation in this cascade caused increased rates of NHEJ in cancer cell (147). 53BP1 also has regulatory roles in AKT, as it was shown that increased expression of 53BP1 was able to significantly reduce the levels of

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AKT in A2780 and HO-8910PM cells while having anti-cancer effects (126).

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53BP1 expression in cancer and its effects on clinical characteristics

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5.1.

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5. Role of 53BP1 in cancer

Because of the aforementioned roles of 53BP1 in DDR cascade, it is unsurprising that this

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molecule has important roles in cancers (148). A study by Djuzenova et al. revealed that the levels

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of 53BP1 were significantly higher in specimens of rectal carcinoma compared to normal controls

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(149). Similar results were shown in skin cancer and cervical cancer specimens (150). Djuzenova et al. also found that lower levels of 53BP1 mRNA were associated with lymph node involvement

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and metastasis (151). Other studies have shown that BP531 could be one of the unique mutations in some cancers, as it is a tumor suppressor. A study by Bouwman et al. revealed that loss of 53BP1

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was a factor contributing to the survival of BRCA-1 deficient breast cells, thereby causing the emergence of triple negative breast cancer cells (67). Squatrito et al. found that 53BP1 was a haploinsufficient tumor suppressor regarding glioma formation, as it was shown to be heterozygously lost in almost 20% of glioblastoma multiforme cells (152). Further evidence

regarding the role of 53BP1 in cancer has emerged in studies examining the relation between the expression of 53BP1 and clinical characteristics of cancer. Jianping et al. found that loss of BP531 was associated with increased tumor size, lower survival and higher stages in colorectal cancers.

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In vitro, silencing 53BP1 resulted in inhibited apoptosis and increased proliferation of HCT-116 cells (153). Neboori et al. found that low 53BP1 was associated with increased local recurrence in breast cancer patients being under therapy with radiotherapy and surgery (14).

5.2.

53BP1 in resistance to treatment and prognosis of treatment

One significance of the status of 53BP1 expression in cancer is its effect on the success of

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therapies(154). Studies have shown that depletion of 53BP1 causes resistance to agents such as

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PARP inhibitors, chemotherapy agents and radiation (155, 156). It also predicts outcomes after

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undergoing treatment. A study by Schouten et al. revealed that BRCA-1 like breast cancer with

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low levels of 53BP1 and high levels of XIST had an inferior response to treatment with high dose

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alkylating agents compared to that with high levels of 53BP1 and low levels of XIST. The

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expression profile of these two proteins contributed significantly to survival and disease free survival after treatment (157). Bonanno et al. in their study investigated the predictive power of

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53BP1 levels in predicting survival in non-small lung cancer cell patients undergoing treatment

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with platinum based agents. They found that patients with low levels of 53BP1 had an overall survival of 19.3 months compared to those with high 53BP1 that had an overall survival of only

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8.2 months (158). Hong et al. found that transfecting ovarian cancer cells, SKOV3, with 53BP1 resulted in decreased migration and proliferation. However, they found that the increase in DNA repair contributed to increased resistance to cisplatin, as the half maximal inhibitory concentration increased to 7.58±0.51 µg/ml from 2.98±0.27 µg/ml (155). Yao et al. in their study evaluated the effect of 53BP1 loss on colorectal cancer cells and found that it led to chemo-resistance towards

5-fluorouracil by inhibiting the ATM-CHK2-P53 pathway. Further, this loss was shown to affect the balance between pro- and anti-apoptotic molecules and reduce the levels of caspases (159). As mentioned before, 53BP1 has direct influence on the function of PARP inhibitors (146, 160).

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Jaspers et al. found that loss of 53BP1 increased resistance to PARP inhibitors, by the reemergence of homologous recombination, in BRCA-1 deficient mouse mammary tumors. They also observed that AZD2461 had the most effect on these cells, compared to other agents (161). Another study showed that 53BP1 loss was the main culprit of the PARP inhibitor resistance in the phosphatase and Tensin homolog (PTEN)-deficient cells such as the glioblastoma cell line

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U251 (162). Hassan et al. suggested that the gene signature of 53BP1 should be used in order to

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select patients for the PARP inhibitor therapy (163). A study by Barazas et al. focused on the role

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of the CTC1-STN1-TEN1 (CST) complex in the PARP inhibitor sensitivity and revealed that loss

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of each of the members of this complex resulted in increased resistance to PARP inhibitors. The

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study suggested that this complex could be a downstream signaling complex of 53BP1/RIF1

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signaling (164). REV7 is another molecule in the downstream signaling of 53BP1. A study by Xu et al. showed that loss of this molecule, which is also known as MAD2L2, led to the restoration of

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HR, via establishing end resection of DSBs which was dependent on CTIP. This molecule is recruited to DSB sites via upstream signaling from the H2AX-MDC1-RNF8-RNF168-53BP1

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pathway. As predictable, disruption of this signaling pathway in any step caused increased

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resistance to PARP inhibitors (165). Regarding the PARP inhibitor sensitivity, it is noteworthy to mention that targeting the upstream signaling of 53BP1 is also an effective method in increasing sensitivity to these agents. For example, a study revealed that disruption of the catalytic activity of ubiquitin-specific peptidase 11 mis-regulated recruitment of 53BP1 and RAD51, causing

increased sensitivity to PARP inhibitors (166). A similar relation was also found between the E3 ubiquitin ligase ring finger protein 168, 53BP1 and PARP inhibitor sensitivity (167).

5.3.

Effect of 53BP1 on angiogenesis

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Formation of new vessels is necessary for tumors to sustain their nutrients and also to proliferate and disseminate through the vascular system. Angiogenesis is dependent on multiple signaling cascades, including HIF-alpha, growth factor and PI3K/AKT signaling. In a study performed by Li X et al., it was shown that 53BP1 had an inverse relation with levels of phosphorylated AKT, which is a regulator of two important pro-angiogenic molecules, MMP9 and MMP2. The

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knockdown of 53BP1 in MCF-7 cells resulted in increased levels of MMP9 and 2, which was

Targeting 53BP1 in cancer

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5.4.

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accompanied with increased angiogenesis in vivo and in vitro (168).

Apart from the diagnostic and prognostic significance of 53BP1, some studies have targeted this

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molecule in cancer in hopes of favorable results. KIM YJ et al. used Glionitrin A, a

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diketopiperazine disulfide on prostate cancer cells of DU145. This agent showed increased cell

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cycle stoppage and increased apoptosis, coupled by the activation of caspase 8, 9 and 3. Further, tumors being treated with this agent had a decreased volume size compared to control tumor

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tissues. Molecular investigations revealed that these effects were mediated by the phosphorylation of 53BP1, which activated DDR transducers such as ATM and ATR and their downstream

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signaling pathway (169). The significant anti-tumor effects of Glionitrin A were previously discovered; however, this study uncovered the molecular basis for its potency for the first time (170). Another study examined the effect of vitamin D and cathepsin L in regulating 53BP1. The study revealed that vitamin D upregulated while cathepsin L downregulated 53BP1, causing increased and decreased DSB repair, respectively (47). The expression of 53BP1 was also targeted

in a study by Siping et al., where long non coding RNAs were used to decrease the expression of the BRAF-activated noncoding RNA (BANCR), which directly affected the expression of CSE1L, a regulator of 53BP1 and other DNA repair proteins (171). Targeting of 53BP1 could also be done

5.5.

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to shift the balance between error prone NHEJ and error free HR.

Utilizing 53BP1 as a marker for DNA damage in cancer

One interesting use of 53BP1 measurement has been in identifying the effect of different therapy regimens on cancer cells with regard to implementing DNA damage in cells. Uehara et al. compared UVB and UVC in pancreatic cancer cell lines MiaPaCa-2, which were implemented in

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skin flaps in mice. They used histocultures and confocal imaging to observe 53BP1 foci and found

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that UVB had a greater penetration, by observing 53BP1 foci in the deeper parts of the tumor

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(172). A similar study was also performed by Miwa S et al., where it was found that UVC induced

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DNA damage within 15 minutes, using a live cell assay of 53BP1 coupled with the green

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fluorescent protein (GFP) (173). 53BP1 was also used as a marker of DNA damage in other cancers

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such as cholangiocarcinoma (174). Another use of detecting 53BP1 is in monitoring DNA repair initiation, specifically non-homologous end joining. An example is a study by Maes et al., in which

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the combined effect of decitabine and JNJ-26481585 was studied on multiple myeloma cells.

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Further, it was shown that RAD51 inhibition led to apoptosis (175). Table 1 summarizes the significance of 53BP1 in various types of cancers not mentioned above.

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6.

Conclusion

We aimed to illustrate the significance of 53BP1 in DDR and DNA repair and also its role in the process of carcinogenesis. It was discussed that 53BP1 was capable of interacting with multiple DNA damage sensors, transducers and effectors, mainly BRCA1. 53BP1 promoted NHEJ by this

means and suppressed the BRCA1 affiliated HR. Further, significant interactions with molecules such as ATM, ATR and CHK were shown. 53BP1 was also able to determine the fate of the cell by regulating functions such as apoptosis, cell cycle arrest and autophagy. It was observed that

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53BP1 impeded cancer by affecting all the aforementioned functions, and that any aberrancy in its function was capable of promoting neoplasia. Additionally, 53BP1 was observed to be a key molecule in determining the prognosis and clinical characteristics of multiple cancers; moreover, some cell lines were observed to gain resistance to treatment by alterations in the gene expression and function of 53BP1. We further discussed that targeting this molecule could be of merit in

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future studies, and illustrated examples to show how targeting the function of 53BP1 increased

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sensitivity to medications and radiotherapy, especially with regard to the status of BRCA1.

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Furthermore, evidence was cited regarding the role of 53BP1 in angiogenesis, a necessary process

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for tumor growth and metastasis. Currently, our knowledge regarding the importance of 53BP1 in cancer is in its primary steps, and future studies can further show how targeting 53BP1 could

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Figure 1: The role of 53BP1 in DNA damage response and its interactions with multiple molecules in the cascade

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Lung

Prostate adenocarcinoma Laryngeal carcinoma

Human specimens (drug and radiation resistant group) Human specimens HEP-2

53BP1 alteration Inactivation

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Sporadic breast cancer specimens MCF-7, MDA-MB231, MDA-MB468, and T47D H520

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Cancer type Cell line cholangiocarcinoma Specimens of patients

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Table 1: The significance of 53BP1 in various types of cancers

Increased activation in nuclear foci Decreased expression Increased ectopic expression

Significance Increased risk of clinically significant recurrence after excision. Activation of 53BP1 marked an increased genomic instability Increased stage and worse prognosis Sensitivity to 5-FU

Ref (176)

(177)

(178) (179)

Decreased signaling Increased expression

Attenuated DNA damage (180) and better radio sensitivity Increased DNA repair caused (181) increased resistance to cisplatin

Mutations in the gene Downregulation by short heparin RNA

Mutations of 53BP1 were seen in prostatic cancer Increased sensitivity to radiotherapy

(182) (183)

B cell lymphoma

Loss of 53BP1

Increased DNA end resection and alteration in translocatome

(184)

Deficiency of 53BP1

Inhibition of radio sensitivity

(185)

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colorectal cancer

activationinduced cytidine deaminase induced HCT116