The MRN complex functions as a sensor of DSBs but also directly in the repair of DNA damage through two major pathways of DNA damage response: homologous recombination repair (HRR) and non-homologous end joining (NHEJ), the latter, error-prone [13]. How exactly the MRN complex participates in the DNA damage response (DDR) has been extensively studied and reviewed in [14]. Briefly, following the DNA damage, the MRN complex is rapidly recruited to DNA damage sites by γ-H2AX and RAD17 [15]. Once bound to DSBs, the MRN complex can recruit and activate various DDR proteins, including ATM [16], and Bloom syndrome protein (BLM) [17]. Next, ATM in turn phosphorylates several targets including MRN complex proteins to start downstream signaling pathways. The MRN complex is required for the activation of ATM as supported by biochemical and genetic analyses in mice, yeast, and human cells [18,19,20,21]. MRN also orchestrates the pathway choice in DSB repair. Indeed, the MRN complex is essential in the formation of single-stranded DNA overhang that serves as a platform for the recruitment of HR repair proteins. First, MRE11 endonuclease and C-terminal binding protein (CtBP)-interacting protein (CtIP) generate an initial resection nick. After that, the exonuclease activities of MRE11 and EXO1/BLM (exonuclease 1/BLM RecQ-like helicase) bidirectionally resect toward and away from the DNA end, which commits to HR and disfavors NHEJ [22]. A recent paper demonstrated that the MRN complex also influences the DSB repair pathway choice by interacting with DNA-dependent protein kinase (DNA-PK). In cases where NHEJ fails due to incompatible DNA ends, DNA-PK promotes DNA end resection by stimulating MRE11 endonuclease activity to remove the complex, triggering further DNA end resection and repair by HR [23].

A major source of endogenous DNA damage in cells is represented by replication stress (RS) [24,25] that, if not correctly handled, may cause genome instability. Cellular response to RS activates checkpoints, primarily mediated by the kinase ATR, to arrest the cell cycle and repair DNA damage. Accumulating evidence demonstrates that the MRN complex entitles several roles in the replication stress response (RSR). The MRN complex can bind DNA structures encountered at stalled or collapsed replication forks (such as ssDNA and dsDNA junctions or breaks); activate ATR; and promote the restart of DNA replication [26,27]. Moreover, MRN-mediated resection of nascent DNA likely frees the replisome from the collapsed RF and generates 3′ overhangs for initiation of HR, which in turn restores the replication under stress [28]. In vitro experiments have shown that MRE11 nuclease activity can process replication structures to form ssDNA gaps behind forks, particularly in the absence of protection from RAD51 [29] or PARP1 and BRCA1 [30], suggesting that the MRN complex may play a role at stalled replication forks also in HR and BER defective cells. Therefore, the MRN complex redistribution to restarting forks prevents the accumulation of chromosomal abnormalities during DNA replication [31].

Additionally, the MRN complex regulates fork progression, altering the protein landscape on nascent DNA by a cGAS-STING-TbK1 pathway-dependent modulation of Interferon stimulated gene 15 (ISG15) [32,33].

Importantly, topoisomerase activity generates DNA breaks as normal intermediates during chromosome metabolism. MRN complex prevents topoisomerase-mediated DSBs by removing TOP1 and TOP2 trapped on DNA [34,35,36,37].

Since DSB accumulation may favor tumor initiation but, at the same time, if strongly stimulated, represents, currently, the main therapeutic strategy to treat tumor cells, it is supposed that MRN complex expression and functionality may impact cancer development and progression as much as therapy effectiveness.

There is accumulating evidence that ATR/ATM-regulated DDR may serve as an inducible barrier to constrain tumor development in its early, pre-invasive stages, by inducing cell death or senescence [38]. As the MRN complex plays a crucial role in ATR and ATM activation in response to RS and DSBs, respectively [18,19,20,21,39], MRN complex inactivation may disturb the ATR/ATM-dependent anti-cancer barrier, leading to cancer progression. Coherently with this idea, patients suffering from ATLD, NBS, and NBSLD are typically characterized by a decrease in ATM activation or activity [12,16], genome instability, and cancer predisposition [40,41].

Importantly, epidemiological and clinical studies have found that heterozygous NBS patients, who are clinically asymptomatic, also display an elevated risk to develop some types of malignant tumors [42], supporting the idea that NBS1 gene is a haploinsufficient oncosuppressor. Moreover, heterozygous NBS patients showed increased frequency of chromosomal translocations [43]. Thus, DDR haploinsufficiency is supposed to be the pathogenic mechanism through which NBS1 heterozygosity predisposes cells to malignancy. Consistently, studies in Nbn^+/− mice showed a significantly increased occurrence of spontaneous solid tumors (affecting the liver, mammary gland, prostate, lung, and lymphocytes) without loss of heterozygosity (LOH), providing a clear relationship between NBS1 heterozygosity and increased cancer risk [44]. Despite studies evaluating the frequency of inherited gene mutations in populations with cancer family history may be considered questionable, due to the potential involvement of other genes segregating with NBS1 locus, other evidence supports the concept that NBS1 is a haploinsufficient oncosuppressor. Indeed, several epidemiological studies report an increased frequency of NBS1 heterozygous germline mutations in patients with sporadic tumors compared to the control population.

Defects in one DNA repair pathway can be compensated for by other DNA repair pathways. Such compensating pathways can be identified in synthetic lethality screens and then specifically targeted for the treatment of DNA repair-defective tumors. Coherently with this idea, several studies support the possibility to use deficiencies in the MRN complex-dependent DDR as an opportunity for therapeutic exploitations in those tumors where the MRN complex is frequently dysfunctional. In vitro experiments demonstrate that deficiency in HR by mutations in the MRN genes may sensitize endometrial [46] and microsatellite unstable colorectal cancer cells [47,48] to treatment with poly (ADP-ribose) polymerase (PARP) inhibitors and might therefore serve as a predictive biomarker of PARP inhibitor therapy. Microsatellite instability-dependent mutations in CtIP and MRE11A confer hypersensitivity to PARP inhibitors in myeloid malignancies [49]. Takagi et al. found SNVs and/or copy number alterations in DDR-associated genes, including MRE11A, in 48.4% of all neuroblastoma analyzed, and these data could explain the high sensitivity to Olaparib observed by the authors in most NB cells, in vitro [50]. Both MRE11 and NBS1 depletion increases sensitivity to PARP-1 inhibitor KU 58948 in breast cancer cell lines [51]. Similarly, MRE11 dysfunction is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3-related (ATR) and PARP but not to the anti-microtubule agent Taxol, supporting that truncated or missense variants of MRE11A may promote hypersensitivity to DNA-damaging therapeutics in breast cancer [52]. Coherently, another study demonstrates that MRE11A and NBS1 transcript levels associate with resistance to Olaparib in breast cancer cell lines [53]. Thus, since MRN defects commonly occur in ER/PR/ERBB2 (triple)-negative breast carcinomas (TNBC), it is expected that PARP inhibitors could be useful in the treatment of this group of patients, presently the most difficult-to-treat subset of breast tumor patients. In Head and neck squamous cell carcinoma (HNSCC), the downregulation of the MRN complex combined with PARPi, leads to accumulation of lethal DNA DSBs in vitro and in vivo [54]. In epithelial ovarian cancer, where a lack of MRN complex protein detection was seen in 41% of the tumors, MRE11 knockdown increased sensitivity towards the PARP inhibitor BMN673 [55]. Moreover, MRE11 inhibition was synthetically lethal in platinum-sensitive XRCC1-deficient ovarian cancer cells and 3D-spheroids [56].

Overexpression or constitutive activation of oncogenes, such as H/KRAS, c-Myc, NMYC, MDM2, BCL-2, and cyclin E, causes alterations of replication timing and progression leading to RS [57]. Therefore, RS is linked to pre-tumor [58,59,60] and tumor [61,62,63,64,65,66,67,68]. Extensive literature supports the concept that cells proliferating under the pressure of oncogenes need to counteract the RS by strengthening the RSR. Accordingly, MRN genes are often overexpressed in tumor tissues [69,70,71,72,73], also because they are transcriptional targets of some oncogenes and proto-oncogenes. In particular, it has been demonstrated that c-Myc transcriptionally regulates NBS1 [74][75]. Coherently with the idea that MRN complex is necessary to counteract oncogene-induced RS, MRE11 inactivation leads to enhanced DNA damage, decreases survival, and increases apoptosis in cells with exogenous overexpression of c-Myc or MYCN [69,73].

Primary anti-cancer therapies, such as ionizing radiation and chemotherapeutic agents, induce cell death by directly or indirectly causing RS and DNA damage. Inherent resistance of tumors to DNA damage often limits the therapeutic efficacy of these agents, becoming the leading cause of treatment failure in cancer patients. Possibly due to the roles of the MRN complex in DDR and RSR [75,76], most studies (with one only exception [70]) support the idea that the MRN complex is a critical factor in driving chemo- and radio-resistance. In particular, nuclear accumulation of the MRN complex was associated with chemo-resistance in gastric cancer [77] and ovarian cancer [56,78], and high expression of RAD50 correlates with radio-resistance in colorectal cancer (CRC) patients [79]. Moreover, in pre-clinic ovarian cancer studies, RAD50 expression resulted higher in platinum-resistant cells [80], and platinum treatment increased nuclear sub-cellular localization of NBS1 [78] and RAD50 [80] more in platinum-resistant cells with respect to the sensitive ones, supporting the idea that accumulation of nuclear MRN proteins could contribute to cisplatin resistance in this tumor background. Coherently, it has been found that MRN targeting may also rescue radioresistance. Indeed, overexpression enhances radio-resistance in non-small cell lung cancer (NSCLC), in vitro [81].

With a similar rationale, other studies showed that MRN complex targeting sensitizes to chemo- and radiotherapy. In particular, RAD50 depletion sensitizes to radiotherapy human nasopharyngeal carcinoma [82] and NSCLC [81] cells. Moreover, the expression of a mutant NBS1 by a dominant negative recombinant adenoviral construct significantly increases cisplatin-induced DNA DSBs and cytotoxicity in HNSCC cell lines [83]. Cells deficient in MRE11 or expressing the nuclease-deficient form of MRE11 resulted in more sensitivity to Etoposide [37]. Moreover, a combination of mirin, an inhibitor for the MRN complex [84], with ionizing radiation treatment significantly enhanced DSBs reduced clonogenic cell survival, inhibited cell proliferation, and promoted cell apoptosis in esophageal squamous cell carcinoma (ESCC) cells [85].

Interestingly, several authors find that cancer stem cells (CSC) from different tissues efficiently resolve RS [86,87,88]. Regarding to this, it has been demonstrated that inhibition of MRE11 and RAD51 effectively kills colorectal CSC resistant to irinotecan and ATR/CHK1 inhibitors via a mitotic catastrophe process, after RSR weakening and defective mitoses [89].

The appearance of dsDNA in the cytoplasm, which is normally a DNA-free environment, triggers potent inflammatory pathways that culminate in the production of interleukin 1β (IL-1β) and type 1 interferon. Such innate immune responses alert the host to the presence of danger and are important for the defense against viruses and bacteria [90].

Upon viral infection, viral DNA in the cytoplasm is perceived as a DSB, triggering endogenous ATM-dependent DNA damage response. The MRN complex has emerged as a detector of many different viral genomes that activates a localized ATM response that specifically prevents viral DNA replication [91]. Importantly, to avoid this response, several viral proteins were found to mediate proteasome-dependent degradation and mislocalization of components of the cellular DNA damage machinery, including the MRN complex [92,93,94,95]. In contrast, MRN was recruited within viral replication compartments and exerted a positive effect on herpes simplex virus 1 (HSV-1) lytic replication [96,97], suggesting that MRN complex could play different roles in Adeno-associated virus (AAV) replication depending on the helper virus that is present.

Recent studies demonstrated that, working as a sensor of cytosolic DNA, the MRN complex has a role in innate immune activation. In particular, cytoplasmic delivery of dsDNA by transfection of DNA or infection with a virus resulted in the formation of distinct dsDNA-RAD50-CARD9 complexes that selectively induced NF-κB signaling for IL-1β production [98]. It was also found that the physical interaction between MRE11 (or RAD50) with dsDNA in the cytoplasm was required for the stimulation of cGAS/STING-inflammatory signaling (IFN genes) [99]. Importantly, NBS1 is not essential for dsDNA-induced type I IFN production [99], but, favoring accumulation of the MRN complex in the nucleus, even decreases cytosolic DNA sensing by MRE11. More recently it has been demonstrated that also NBS1 binds to micronuclear DNA, probably via MDC1, and recruits ATM and CtIP to convert micronuclear dsDNA ends to ssDNA ends. This conversion prevents cGAS from binding to micronuclear DNA and avoids the cGAS-STING dependent induction of immune signaling and cellular senescence [100]. Therefore, NBS1 negatively regulates the MRE11-RAD50-dependent production of IFN genes both by the transport of MRE11 in the nucleus and by reducing dsDNA in the cytoplasm.

Programmed DSBs and repair systems such as V(D)J and class-switch recombination (CSR) are part of a developmental program that ensures the diversity of antigen receptors in B and T lymphocytes and the production of specific classes of immunoglobulin. Inability to persecute the programmed DDR results in the primary immunodeficiency (PID) [101]. Much evidence supports the role of NBS1 in both V(D)J [102] and CS recombination [103]. NBS1 localizes at switch regions in activated B cells [104] and Nbn conditional KO mice show impaired class-switch recombination [105]. Coherently, NBS patients have reduced titers of switched serum isotypes and altered switch junctions [106]. Interestingly, immunodeficiency is a hallmark of NBS but not of NBSLD and AT-LD patients. This aspect may suggest an MRN complex independent role of NBS1 in lymphoid cells.

Many DDR proteins, particularly those involved in responding to DNA DSBs, physically associate with telomeres and play key roles in their maintenance. In fact, components of the DDR are necessary both for normal telomere homeostasis and for responding to dysfunctional telomeres [107]. The MRN complex associates with human telomeres through interaction with TRF2 and it is required for the activation of ATM at dysfunctional telomeres [108,109]. Moreover, the MRN complex participates in the formation of the structure of t-loops, the lariats formed through the strand invasion of the telomere terminus into the duplex telomeric DNA, which contributes to telomere protection, and consequently to the preservation of genome stability, by effectively shielding the chromosome ends from DNA damage response factors that interact with DNA ends [107,109]. Telomeres of telomerase-negative primary cells recruit MRE11, phosphorylated NBS1, and ATM in every G2 phase of the cell cycle, when telomeres are unprotected and accessible to modifying enzymes. Degradation of the MRN complex, as well as inhibition of ATM, led to telomere dysfunction. Therefore, a localized MRN complex-dependent DDR at telomeres after replication is essential for recruiting the processing machinery that promotes the formation of a chromosome end protection complex [110], avoiding chromosome instability and senescence.

The accurate chromosomal segregation that occurs during cell division is controlled by the mitotic spindle and alterations in centrosome stability/number or mistakes in spindle formation/disassembly can result in aneuploidy or cytokinesis failure, which are linked to cancer [111,112]. Importantly, the MRN complex seems to be associated with both centrosome and mitotic spindle homeostasis. In particular, NBS1 localizes at the centrosome in mitosis and in interphase, and because its depletion induces centrosome hyper-duplication, it is widely accepted that its expression is essential for correct centrosome duplication [113]. Interestingly, MRE11 localizes both at centrosome and microtubules in mitotic cells [114], and its depletion triggers centrosome amplification [115]. Moreover, it was demonstrated that the MRN complex is necessary for RCC1-mediated RanGTP gradient, essential for the self-assembly of microtubules into a bipolar structure. Indeed, recent studies found that the inhibition of MRN complex function in mammalian cells, reducing RCC1 association with mitotic chromosomes, disrupting the RCC1-dependent RanGTP gradient, and triggering metaphase delay [116]. Moreover, Xu et al. found that the MRN complex together with MMAP (which forms a mitosis-specific complex named mMRN) regulates the PLK1–KIF2A signaling cascade, widely known for being involved in the microtubule depolarization and consequently in spindly disassembly [114]. Curiously, centrioles and centrosomes contain RNA but do not contain any DNA [117]; therefore, it is possible that the MRN complex regulates centrosome/spindle stability in a DDR-independent manner.

Numerous studies indicate both inverse and positive correlations between MRN complex and mitogenic pathways expression. In particular, several authors found that NBS1 expression negatively regulates the NOTCH pathway. It was demonstrated that, independently of the DDR, NBS1 deletion up-regulates the NICD protein level, as well as NOTCH activity in neurons, causing in turn repression of neurite outgrowth and neuronal migration in post-mitotic neurons [118]. On the contrary, NBS1 expression positively regulates RAS/RAF/MEK/ERK cascade upon activation by the growth hormone IGF-1 [121]. Despite a single exception in ESCC, where NBS1 expression inversely correlates with SNAIL expression and reduces E-cadherin expression [122], increasing literature supports a positive correlation between MRN complex expression and invasion/metastasis-related genes. Cristina Espinosa-Diez et al. demonstrated that in the presence of significant DNA damage, MRN complex targeting by miR-494 and miR99b decreases VEGF signaling and thereby angiogenesis, thus supporting the angiogenic role of the MRN complex in endothelial cells [123]. In papillary thyroid carcinoma (PTC), the downregulation of MRE11 and RAD50 expression, through the lncRNA SLC26A4-AS1-mediated disruption of the DDX5-E2F1 transcription factor complex, inhibits the invasion and metastasis capability of cancer cells [124]. In oral cancer, it was found that MRE11 promotes proliferation, epithelial–mesenchymal transition, and metastasis regulating RUNX2, CXCR4, AKT, and FOXA2 in a nuclease-independent manner [125]. Moreover, in breast cancer, MRE11 overexpression promotes proliferation by activation of the STAT3 signaling and its downstream effectors on one side, and tumor cell invasion and migration by an increase in secretion of metastasis-associated MMP proteins (MMP-2 and MMP-9), on the other side [126]. Overexpression of NBS1 induces EMT through the upregulation of the PI3-KINASE/AKT/SNAIL/MMP2 axis in HNSCC [127] and by MMP-2-independent expression of two heat shock proteins HSPA4 and HSPA14 in NSCLC [128]. In high-grade serous ovarian cancer, RAD50 overexpression activates NF-kB which increases several mesenchymal phenotype markers such as N-cadherin, Vimentin, SNAIL, and TWIST, and reduces the expression of epithelial phenotype marker E-cadherin [129]. Analysis of differentially expressed genes associated with low expression of MRE11A, RAD50, and NBS1 identified an increased expression of genes associated with mitochondrial dysfunction and metabolic reprogramming that could contribute to the aggressive behavior of MRN-deficient tumors [130]. Importantly, cancer-related pathways are the most enriched pathways that were found in NBS-fibroblasts compared to healthy fibroblasts, as well as in NBS-iPSCs compared to embryonic stem cells [131].