repair pathways in cancer.

Review

Targeting the microRNA-regulating DNA damage/repair pathways in cancer 1.

Introduction

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The DNA damage response

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miRNA biogenesis: expression

Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by University of Guelph on 09/29/14 For personal use only.

and maturation 4.

DNA damage-regulated miRNAs

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MiRNA-mediated regulation of DDR genes

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Role of the DDR-miRNAs in cancer tumorigenesis and progression

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miRNA-DDR and chemotherapy sensitivity

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DDR-related miRNAs as predictive biomarkers in the clinical setting

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Conclusion Expert opinion

Giulia Bottai, Barbara Pasculli, George A Calin & Libero Santarpia† †

IRCCS Humanitas Clinical and Research Institute, Experimental Therapeutics Unit, Milan, Italy

Introduction: Maintenance of genome stability requires the integrity of the DNA repair machinery. DNA damage response (DDR) determines cell fate and regulates the expression of microRNAs (miRNAs), which in turn may also regulate important components of the DNA repair machinery. Areas covered: In this review, we describe the bidirectional connection between miRNAs and DDR and their link with important biological functions such as, DNA repair, cell cycle and apoptosis in cancer. Furthermore, we highlight the potential implications of recent findings on miRNA/DDR in determining chemotherapy response in cancer patients, and the use of these biomarkers for novel potential therapeutic approaches. Expert opinion: Defects in the DDR and deregulation of miRNAs are important hallmarks of human cancer. A full understanding of the mechanisms underlying the connection between miRNAs and DDR/DNA repair pathways will positively impact our knowledge on human tumor biology and on different responses to distinct drugs. Specific miRNAs interact with distinct DDR components and are promising targets for enhancing the effects of, and/or to overcome the resistance to, conventional chemotherapeutic agents. Finally, the development of innovative tools to deliver miRNA-targeting oligonucleotides may represents novel types of cancer interventions in clinic. Keywords: apoptosis, cancer therapies, cell cycle, chemotherapy, DNA damage response, DNA repair, drug response, microRNAs Expert Opin. Biol. Ther. [Early Online]

1.

Introduction

DNA repair machinery maintains genomic integrity and is frequently altered in several types of human cancer [1-3]. In order to preserve the integrity of the genome, cells trigger a specific cellular response, referred to as DNA damage response (DDR), which includes several distinct DNA repair pathways. The DNA repair pathways allow normal cells to repair DNA damage or induce apoptosis and cell cycle arrest if repair is not possible [4]. Disruption of these pathways in cancer leads to an increase in genomic instability and mutagenesis. Anticancer drugs are highly influenced by the cellular DNA repair capacity of neoplastic cells, and specific alterations in DNA repair networks have been reported to determine different chemotherapy responses in cancer patients [5]. Several studies suggested that microRNAs (miRNAs) play a key role in the regulation of the DDR. [6,7]. MiRNAs are an evolutionarily conserved group of small (18 -- 25 nucleotides) endogenous noncoding RNAs (ncRNAs) that regulate gene expression by repressing translation or promoting the degradation of their target mRNAs. These single-stranded RNA molecules regulate the stability and translation of their target mRNAs by perfect or imperfect base pairing at the 3¢ untranslated region (3¢ UTR) of the mRNA. The conserved heptametrical sequence situated at positions 2 -- 7 from the miRNA 5¢ end, known as ‘seed sequence’, is essential for 10.1517/14712598.2014.950650 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. . .

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DNA repair machinery maintains genomic integrity and is frequently altered in human cancer. DNA damage response (DDR) is a potent regulator of microRNA (miRNA) expression, and miRNA-mediated gene silencing has been shown to modulate the activity of DNA repair pathways. Chemotherapy induces the activation of several components of the DNA repair machinery, which is regulated by a core of specific miRNAs. Targeting DDR-responsive miRNAs can overcome resistance to chemotherapy, enhancing its cytotoxic effects. MiRNAs are potential predictors of response to different chemotherapy regimens and targeted-therapies.

This box summarizes key points contained in the article.

recognition and binding of the miRNA to the mRNA [8]. In the human genome, around 40% of genes are expected to be targeted by miRNAs [8]. To date, miRNAs have been assigned a regulatory role in a variety of physiological and pathological processes, including cancer [9]. Furthermore, the DDR and DNA damage are known regulator of miRNA expression [10,11]. Several studies have shown that the DDRmiRNA network could affect cellular sensitivity to chemotherapeutic drugs [12]. Despite the continuous development of novel targeted therapies, chemotherapy remains the main treatment option for many types of cancers, especially in advanced and/or metastatic settings. However, drug resistance and side effects related to chemotherapy may significantly limit the effectiveness of such treatments. Therefore, the proper selection of patients and the need to overcome drug resistance remain a substantial challenge for a better and personalized clinical management [13]. Future work is needed to address the role of DNA damage responsive miRNAs and their targets in the DDR/DNA repair pathways. A deep knowledge of mechanisms underlying the DDR-miRNA network will most likely lead to novel strategies able to overcome resistance to chemotherapy, enhancing drug effects on cancer cells and minimizing side effects in clinical practice. 2.

The DNA damage response

The human genome is subject to constant damage through a combination of endogenous and exogenous genotoxic stresses. DNA damage may result from infidelities of DNA or from endogenously produced reactive oxygen species (ROS). In addition, several extrinsic factors, such as ultraviolet (UV) light, ionizing radiation (IR), chemo- and radio-therapeutic agents may damage DNA [14]. When not repaired properly, such damages may lead to mutations, deletions, insertions or chromosomal rearrangements upon DNA replication or cell division. To maintain the stability of the genome, cells trigger a specific network of cellular responses. The DDR is 2

conserved across all organisms and is designed to detect DNA damage and activate the most appropriate signaling pathway to mediate its repair [2]. The DDR encompasses DNA repair mechanisms, cell cycle control pathways, replication bypass mechanisms and apoptosis and is finely regulated by a variety of proteins, which function as sensors, mediators, transducers and effectors. The information collected and conveyed by these proteins will finally be used in determining cell fate, either by arresting the cell cycle to allow repair of damaged DNA or, when the damage is irreparable, by initiating programs of cell death. Five main repair pathways have been described: the nucleotide excision repair (NER) pathway, base excision repair (BER) pathway, mismatch repair (MMR) pathway and the two double-strand breaks (DSB) repair pathways, non-homologous end-joining (NHEJ) and homology-directed repair (HDR; also known as homologous recombination). NER mostly deals with larger, helix-distorting lesions that result from chemical modifications of DNA bases upon exposure to environmental mutagens, such as UV light, ROS, radiation and chemotherapeutic agents. BER corrects damage to a single base caused by ROS-induced methylation, oxidation, alkylation, hydrolysis or deamination. The MMR pathway is responsible for repairing replication errors that escape processing by the 3¢-5¢ proofreading exonuclease activity of DNA polymerases. DSBs are repaired by NHEJ in all phases of cell cycle but mainly before replication, in the absence of an identical copy of DNA, whereas during DNA replication DSBs can be mended by HDR. The knowledge of the physiological roles and the mechanisms of DNA repair pathways is important for understanding how their deficiencies or abnormalities could affect the development of numerous diseases. In fact, defects in DNA damage signaling or repair has been associated with several human diseases, cancer risk and therapy outcomes [3-5].

miRNA biogenesis: expression and maturation

3.

The biogenesis of miRNAs comprises several steps, including transcription, processing/maturation and degradation (Figure 1). Depending on their genomic location, miRNA genes can be transcribed from two different pathways. Intergenic miRNAs are transcribed as primary miRNAs (pri-miRNAs) by RNA polymerase II, as they contain their own promoter and regulatory sequences, whereas intronic miRNAs are co-transcribed with their host genes from a common promoter [8]. Following transcription, the pri-miRNAs from intergenic miRNAs are 5¢ capped, 3¢ polyadenylated and cleaved in the nucleus by Drosha/Pasha microprocessor, whereas intronic miRNAs are directly cleaved without affecting the splicing of host genes [8]. This first cleavage step generates an approximately 70-nucleotide hairpin-shaped precursor, called pre-miRNA, which contains 25 -- 30 base pair stems and relatively small loops with 3¢ overhangs [8]. Subsequently, a RanGTP-binding nuclear

Expert Opin. Biol. Ther. (2014) 14(11)

Targeting the microRNA-regulating DNA damage/repair pathways in cancer

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Figure 1. Regulation of miRNA biogenesis by the DNA damage response. Once the cell senses DNA damage, the major transducer of DDR, ATM, transmits DNA damage signals to the miRNA biosynthetic pathway through specific proteins, such as TP53 and KSRP. TP53 tumor-suppressor family members participate in the regulation of miRNA expression at both the transcriptional and post-transcriptional levels. As a transcription factor, TP53 increases or decreases the transcription of several miRNAs. TP53 is linked to Drosha/Pasha complex through direct interaction with p68/p72, which enhances processing of pri-miRNAs to pre-miRNAs. TP63 (*), induces transcription of Dicer and miRNA-130b. ATM-mediated phosphorylation of KSRP promotes its interaction with a subset of miRNA precursors and increases the processing of target pri-miRNAs to premiRNAs by the Drosha/Pasha complex. DDR: DNA damage response; miRNA: MicroRNA; pri-miRNAs: Primary miRNAs; RISC: RNA-induced silencing complex.


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transporter, exportin-5, mediates the pre-miRNA translocation from the nucleus to the cytoplasm. In the cytoplasm, the endoribonuclease Dicer, a member of the RNase III family, and its double-stranded RNA binding cofactor TRBP, cleave premiRNAs into a transient duplex of 20 -- 25 nucleotide, consisting of a functional miRNA strand, the guide strand, and a passenger strand [15]. Alternatively, it was proposed an additional endonuclease step in which Ago2, a member of the Argonaute family (Ago1-4), cleaves the pre-miRNA, generating a nicked Ago2-cleaved miRNA precursor (ac-pre-miRNA hairpin) that serves as Dicer substrate [16]. Only one strand (the guide strand) of the miRNA duplex is selected on the basis of the stability of the 5¢ end, whereas the other strand (the passenger strand) is usually degraded. Considering that the ac-premiRNA harbors a single strand cleavage in the 3¢ arm of the pre-miRNA, this could contribute to the strand selection process and facilitate removal of the passenger strand and unwinding of the miRNA duplex after Dicer cleavage [16]. At this stage, the mature miRNA is loaded into an RNA-induced silencing complex (RISC) in which one of the four Argonaute proteins mediates the binding between miRNA and target mRNA [16]. Degradation of the target mRNA occurs only when miRNA and its target mRNA are perfectly, or nearly perfectly, complementary to each other. This process is the same of the RNA interference induced by artificial small interfering RNAs. When there is only partial sequence-complementarity, translational repression occurs. Even though the mature miRNA loaded into RISC complex is protected from degradation by Ago proteins, after finishing its task, it will be degraded by the 5¢-3¢ exoribonuclease XRN2 or by other mechanisms [8,17]. 4.

DNA damage-regulated miRNAs

Many evidences suggest that there is a bidirectional connection between miRNAs and the DDR. The DDR is a known regulator of miRNA expression at both transcriptional and post-transcriptional levels (Figure 1), and miRNA-mediated gene silencing has been shown to modulate the activity of the DDR (Figure 2) [6,7,18]. Treatment with different types of chemotherapeutic agents has been shown to result in differential expression profiles of miRNAs [5,19]. Depending on DNA damage type and level, a unique set of miRNAs as well as a common core miRNA signature are activated, suggesting that miRNAs regulate the DDR by mechanisms based on the type and/or the intensity of DNA damage [18]. Transcriptional regulation of miRNAs after DNA damage

4.1

DNA damage can regulate miRNAs expression at the transcriptional level (Figure 1). Many miRNA promoters have characteristics similar to those of normal protein-coding genes and are controlled by several transcription factors, such as TP53, E2F and MYC. Transcription factors can regulate miRNAs expression by directly binding to miRNA promoters and modulating their transcriptional activity, or by modifying 4

the expression of miRNA processing machinery components. Genome-wide miRNA screening for TP53-dependent regulation following DNA damage and miRNA expression profiles of wild-type and TP53-deficient cells have been used to identify miRNAs involved in TP53-mediated transcriptional pathways. The miRNA-34 family (miRNA-34a-c), which is induced by DNA damage and oncogenic stress, has been identified as a direct transcriptional target of the tumor suppressor TP53 [20]. MiRNA-34a is encoded by an individual transcript and has an almost ubiquitary pattern of expression, whereas miRNA-34b and miRNA-34c share a common primary transcript and are mainly expressed in lung tissue. TP53 directly binds to miRNA-34 family promoters and activate their transcription. In turn, miRNA-34 family members have been shown to repress the mRNA transcripts of several genes involved in the regulation of cell cycle, cell proliferation and survival, such as BCL2, CCND1 CCNE2, MYC, CDK4, CDK6 and SIRT1 [20]. Inactivation of endogenous miRNA-34a strongly inhibits TP53-mediated apoptosis, whereas its ectopic expression promotes TP53-mediated apoptosis, cell cycle arrest or senescence [20]. Most of the targets of miRNA-34a have a role in cell cycle progression, apoptosis and DNA repair. Interestingly, miRNA-34a through targeting SIRT1 may form a positive feedback loop by increasing the acetylation of TP53, which in turn induces the expression of its transcriptional targets, such as P21 and PUMA, regulating cell cycle and apoptosis, respectively [20]. Although miRNA-34c is induced by TP53 following DNA damage, in the absence of TP53 its induction still occurs, suggesting the existence of an alternative pathway that likely involves p38 MAPK signaling [21]. The overexpression of miRNA-34c decreases MYC expression, whereas its inhibition prevents the DNA damage-induced cell cycle arrest and results in an increased DNA synthesis [21]. These data suggest that miRNA-34c induction by DNA damage allows the TP53or the p38 MAPK-dependent suppression of MYC and decreases the rate of improper replication to maintain genomic integrity. DNA damage promotes the TP53-dependent upregulation of miRNA-192, miRNA-194 and miRNA-215. The genomic region surrounding the miRNA-194/miRNA-215 cluster contains a putative TP53-binding element, indicating that these miRNAs are transcriptionally activated by TP53 [20]. The expression of miRNA-192 and miRNA-215 induces cell cycle arrest and targets several transcripts involved in cell cycle checkpoints [22]. In TP53+/+ colorectal carcinoma cell line, but not in TP53-/- cells, miRNA-192 was found to increase the level of P21, indicating the existence of a positive feedback loop for the regulation of TP53 activity [22]. Furthermore, miRNA-192 and miRNA-215 are downregulated in many colon cancer samples, suggesting a role as tumor suppressor miRNAs [22]. The miRNA-17-92 cluster is repressed under hypoxic conditions via a TP53-dependent mechanism, resulting in sensitization to hypoxia-induced apoptosis [23]. The TP53-mediated miRNA-17-92 repression is modulated by



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DNA-PK Ku70 Ku80 XRCC4 LIG4 ERCC1 CtIP BRCA1 BRCA2 FANCD2 RAD51 RAD52 RAD23B

miR-99 miR-182 miR-146 miR-28 Let-7

miR-210 miR-373

Figure 2. MicroRNAs regulate the DNA damage response through modulating key components of various pathways. The core proteins during the DDR pathway are summarized in the order of their presentation in the text (sensors and mediators, transducers and effectors of DNA repair, cell-cycle checkpoint and apoptosis). DDR: DNA damage response; ERCC1: Excision repair cross-complementation group 1; MRN: MRE11/RAD50/NSB1; PLK1: Polo-like kinase 1.

preventing the TATA-binding protein transcriptional factor from binding to a TATA box that overlaps with the TP53binding site within the miRNA-17-92 promoter. TP53 also induces miRNA-145 transcription by directly binding to its promoter, and miRNA-145 directly targets MYC, suggesting that TP53 may repress MYC through induction of miRNA145 [19]. It was reported that DNA damage increases the miRNA-29 family expression in a TP53-dependent manner [24]. MiRNA-29 represses WIP1 phosphatase, an important component of the DDR that inhibits the activation and the stabilization of TP53, ultimately leading to TP53 induction [24]. Furthermore, the full-length isoform of TP63, which belongs to the TP53 family of transcription factors, has an important role in suppressing tumorigenesis and metastasis. TP63, induced by DNA damage or other stresses, directly binds the Dicer promoter and activates its transcription, as

well as that of miRNA-130b. The modulation of Dicer and miRNA-130b expression markedly affects the metastatic potential of cells lacking TP63 [25]. Two other transcription factors involved in DNA damageinduced cell cycle checkpoints, MYC and E2F, regulate the expression of several miRNAs. Both factors induce transcription of the miRNA-17-92 cluster that forms a feedback loop by inhibiting E2F expression [26]. E2F transcription factors are repressed by several other miRNAs, including miRNA-106a-92 and miRNA-106b-25 cluster members, miRNA-210, miRNA-128, miRNA-34 and miRNA-20 [27]. Regulation of miRNA processing and maturation by DNA damage

4.2

DNA damage can also regulate miRNA expression by modulating the miRNA processing and maturation steps (Figure 1).


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Several miRNAs, including miRNA-16-1, miRNA-143 and miRNA-145, have been found to be upregulated by DNA damage [28]. TP53 interacts with the Drosha/Pasha processing complex through the association with the RNA helicase p68 and facilitates the processing of pri-miRNAs to pre-miRNAs. Transcriptionally inactive TP53 mutants interfere with the functional assembly between Drosha/Pasha complex and p68, resulting in the attenuation of miRNA processing activity [28]. Most TP53 mutations found in cancers are located in a domain required for miRNA processing and transcriptional activity [28]. Thus, loss of TP53 functions in miRNAs transcription and processing might contribute to cancer progression. DNA damage triggers a cell-cycle-dependent relocalization of Ago2 into stress granules and promotes the expression of several miRNAs, including miRNA-16 [29]. The stress granules regulate mRNA metabolism and impair translation of many housekeeping mRNAs during stress. The findings that Ago2 relocalizes to stress granules and that miRNA-16 downregulates the cell cycle checkpoint gene CDC25A and WIP1 phosphatase early after DNA damage, support the idea that miRNA-mediated gene silencing operates earlier than most gene transcriptional responses and that this process is an integral part of the DDR [29]. A complex network of transcriptional and posttranscriptional regulation has been analyzed by comparative and computational genomic approaches, showing that TP63 and TP73, together with TP53, could function as both positive and negative regulators of miRNA processing and maturation. Indeed, TP53/TP63/TP73 axis regulates the processing of several miRNAs, including let-7, miRNA-21, miRNA-16, miRNA-107, miRNA-134, miRNA-143, miRNA-145, miRNA-200c, miRNA-449a and miRNA-503 [30]. In addition to direct interaction with the miRNA processing complex, other mechanisms have been suggested by which TP53/TP63/ TP73 can regulate miRNA processing. TP53-modulated miRNAs could target most of the components of the miRNA processing/maturation complex, such as Drosha/Pasha, Dicer/ TRBP and Ago proteins, and promoters of several miRNA processing factors, such as Dicer, contain TP53-responsive elements, suggesting that they could be direct transcriptional targets of TP53/TP63/TP73. Thus, a feedback effect could help maintain physiological levels of miRNAs [20]. In addition to its role in transcriptional miRNA regulation, MYC was also recently shown to promote miRNA processing by upregulating the Drosha expression level, revealing a previously unrecognized function of MYC in miRNA processing as valuable insight into a new aspect of how MYC regulates miRNA expression [31]. The MLH1-PMS2 heterodimer, involved in the MMR pathway, was found to positively regulate the processing of several miRNAs, including miRNA-422a, by binding to pri-miRNAs, interacting with the Drosha/Pasha complex, and stimulating the Drosha/Pasha-mediated processing of pri-miRNAs to pre-miRNAs [32]. Furthermore, the tumor suppressor BRCA, a key component of the DSBs response, 6

was recently shown to increase the expression of both precursor and mature forms of let-7a-1, miRNA-16-1, miRNA145 and miRNA-34a by direct association with Drosha and p68 RNA helicase [33]. These results suggest that MMR components and BRCA1 regulate miRNA biogenesis via the Drosha microprocessor complex and this novel function may be linked to tumor suppressor mechanism and maintenance of genomic stability. Another key signaling component of the DDR, ATM, was found to be directly involved in miRNA processing. ATM and its relative ATR are serine/threonine kinases that transduce the DNA damage signals to downstream effectors. These kinases regulate several cellular processes including DNA repair, cell cycle and apoptosis, by phosphorylating a wide range of target proteins. Following the induction of DNA DSBs, several miRNAs are significantly induced through ATM activation, whereas a small group of miRNAs is consistently reduced [34]. Among these miRNAs, a set of miRNAs was found to be associated with the RNA-binding protein KSRP [35]. KSRP is a multifunctional, AU-rich single-strand RNA-binding protein that regulates RNA splicing, localization and degradation. KSRP was reported to interact with guanosine-rich regions, including GGG triplets in the terminal loop of pre-miRNA precursors, and to serve as a component of both Drosha and Dicer complexes [35]. Thus, KSRP positively regulates the maturation of a cohort of miRNA precursors, including pri-miRNA-1, pri-miRNA-15, primiRNA-21 and pri-let-7 (Figure 1) [35]. ATM-dependent phosphorylation of KSRP significantly enhances the recruitment of target pri-miRNAs to the Drosha complex and increases their processing, whereas mutations in the ATMdependent phosphorylation sites on KSRP impaires its miRNA-regulating activity [34]. Considering that some miRNAs are reduced after DNA damage in an ATM-dependent manner, ATM could be also involved in inhibitory pathways that downregulate miRNA expression [29]. These findings support the existence of a critical link between the DDR and miRNA processing pathway in which ATM functions as a key regulator of KSRP activity. KSRP acts in turn as a molecular gatekeeper to promote the expression of a specific cohort of miRNAs involved in the DDR. It was also shown that nuclear export of pre-miRNAs through exportin-5 is enhanced after DNA damage in an ATM-dependent manner, defining an important role of DNA-damage signaling in miRNA transport and maturation [36]. ATM-independent regulation may also connect DNA damage signaling to the miRNA pathway [18]. In addition to ATM, many kinases are activated following DNA damage. ERKmediated phosphorylation of TRBP in the Dicer complex has been shown to stabilize the complex itself and to increase the expression of mature miRNAs. The upregulation of several miRNAs, such as miRNA-17, miRNA-20a and miRNA-92a, is promoted by the activated form of TRBP, whereas the let7 tumor suppressor family was found to be downregulated, suggesting a definite TRBP activation-dependent miRNA



expression pattern [37]. Taken together, DNA damage can induce the expression of specific miRNAs and modulate their processing. Further studies are needed to understand whether DNA damage may influence miRNA expression through the regulation of miRNA degradation.


5.

MiRNA-mediated regulation of DDR genes

In addition to being regulated by canonical signaling pathways at the transcriptional and post-translation levels, the DDR components are also post-transcriptional regulated. MiRNAs, as endogenous gene regulators that affect protein stability, offer an important degree of regulation for DDRs, and it is becoming clear that DNA damage responsive genes are subjected to modulation by miRNAs. The principal cross-talks between miRNAs and DDR are shown in Figure 2. Sensors and mediators of DNA damage signaling The MRE11/RAD50/NSB1 (MRN) complex acts as a sensor of DSB to recruit ATM. Following DNA damage, the histone variant H2AX is extensively phosphorylated by ATM and ATR kinases. This post-translational modification represents one of the earliest events in the DDR and is essential for the subsequent recruitment of several checkpoints and DNA repair proteins to the damage site. The protein mediator of DNA-damage checkpoint 1 binds to phospho-H2AX and to MRN complex in order to recruit the ubiquitin ligase RNF8 and to form a scaffold that facilitates the accumulation of RNF168, BRCA1 and 53BP1 around DNA lesions [38]. MiRNA-24 was identified by miRNA arrays in terminally differentiated hematopoietic cells. Overexpression of miRNA-24 inhibited H2AX expression and DNA repair and increased the sensitivity to g-irradiation and genotoxic agents [39]. Both miRNA-138 and miRNA-542-3p were shown to reduce phospho-H2AX foci formation after DNA damage [40] and overexpression of miRNA-138 was found to inhibit HDR and increase cellular sensitivity to DNA damaging agents such as cisplatin and camptothecin [40]. Furthermore, ncRNAs, which are Drosha- and Dicerdependent RNA products with the sequence of the damaged site, were recently found to activate the DDR following DNA damage in a MRN-dependent manner [41]. Although these RNAs act differently from canonical miRNAs, they add another layer of complexity to the ncRNAs-mediated control of DDR activation at sites of DNA damage. The MMR pathway is responsible for correcting mismatched nucleotides that may arise from polymerase misincorporation errors, recombination between heteroallelic parental chromosomes, or chemical and physical damage to the DNA. Inactivation of MMR is one of the principal causes of genomic instability [42]. The overexpression of miRNA-155 was shown to significantly downregulate the MMR proteins MSH2, MSH6 and MLH1, inducing microsatellite instability (MSI) in human colorectal cancer, providing support for miRNA-155 modulation of MMR as a mechanism of cancer pathogenesis [42]. 5.1

Transducers of the DDR In response to DSB DNA damage, ATM becomes activated by autophosphorylation on serine residues and the homeostatic regulation of ATM activity in the DDR is primarily mediated by the WIP1 phosphatase. A recent examination of miRNA-binding motifs in the 3¢ UTR region of ATM led to the identification of miRNA-421 as a regulator of ATM. Ectopic expression of miRNA-421 results in a defective S-phase cell cycle checkpoint and in an increased radiosensitivity [30]. In both cell lines and patient’s tissue samples of breast and colorectal cancer, miRNA-18a was found to be upregulated. Ectopic expression of miRNA-18a leads to a downregulation of ATM by directly targeting the ATM-3¢ UTR region. Inhibition of miRNA-18a results in an increase of DNA damage repair and of HR efficiency and a reduced cellular radiosensitivity [10,43]. The oncogenic miRNA-27a, which is overexpressed in several tumors, directly regulated ATM, promoting cell proliferation of non-irradiated and irradiated lung adenocarcinoma cells and affecting DBS rejoining kinetics after irradiation [44]. MiRNA-181a/b expression was found to be associated with aggressive breast cancers and to inversely correlate with ATM levels [45]. ATM is also a validated target of miRNA-100 and miRNA-101, and overexpression of these miRNAs correlates with aberrant DNA repair and enhanced radiosensitivity in vitro and in vivo [43]. These miRNAs-ATM pathways most likely contribute to the DDR in a variety of ways considering the many targets of transphosphorylation of ATM. These results suggest that these miRNAs might be novel therapeutic targets and could be helpful in tailoring more effective treatments. Compared with ATM, which is primarily activated by DNA DSBs, ATR responds to a broader spectrum of DNA damage and replication interference, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) adjacent to ssDNA, adducts, cross-links and inhibition of DNA polymerase. MiRNA-185 was found to negatively regulate ATR expression at post-transcriptional level, to enhance radiationinduced apoptosis and to inhibit proliferation, indicating a potential application of this miRNA as a tool to increase the sensitivity of tumor cells to radiotherapy [43]. 5.2

Effectors of DNA repair NHEJ and HDR represent two major DSB repair pathways, which occur in different phases of the cell cycle. The majority of DSBs are repaired by NHEJ in G1-phase of the cell cycle and the core proteins required for NHEJ include Artemis, DNA ligase IV (LIG4), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku70, Ku80, XRCC4 and XLF/ Cernunnos. The Ku70/80 heterodimer binds to DSB ends, recruits DNA-PKcs and consequently coordinates the end processing with rejoining by recruiting XRCC4, XLF and LIG4 [46]. DSBs can be also repaired by HDR. CtIP-BRCA1 complex mediates resection of a DSB to produce 3¢ ssDNA overhangs [46], and RAD52 and RAD51 paralogs (-A, -B, -C) 5.3


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then promote strand invasion and displacement. The resynthesis of the broken DNA sequence, using the intact sister chromatids as templates, is then mediated by RAD54 and the RAD51-BRCA2 complex. MiRNA-210 and miRNA-373 are upregulated by hypoxia-inducible factor 1a in hypoxic conditions [47]. The overexpression of miRNA-210 was found to inhibit RAD52 expression, whereas the overexpression of miRNA-373 decreased the NER protein, RAD23B, as well as the RAD52 levels. Consistently, both RAD23B and RAD52 are downregulated during hypoxia. These data suggest that these hypoxia-inducible miRNAs could influence the effects of hypoxia on DDR in cancer, by modulating the expression of HDR- and NER-related proteins [48]. Other miRNAs were found to regulate the expression of key proteins c of the DDR pathways, such as miRNA-101 targeting DNAPK, miRNA-335 suppressing CtIP and let-7 family targeting BRCA1, BRCA2, FANCD2 and CHEK1 [42,49,50]. BRCA1, a tumor suppressor gene, encodes a nuclear phosphoprotein that plays a key role in maintaining genomic stability and mutations in BRCA1 are associated with an increased risk of developing breast and ovarian cancer [51]. BRCA1, beside other tumor suppressors, DNA-damage sensors and transducers, forms a large multisubunit protein complex that is recruited to DNA lesions facilitating DNA repair and cell-cycle regulation [51]. MiRNA-182 targets several genes involved in DNA repair, including BRCA1 [43]. Antagonizing miRNA-182 enhances BRCA1 protein levels and protects breast tumor cells from IR-induced cell death, while overexpressing miRNA-182 in breast tumor cells reduces BRCA1 protein, impairs HDR pathway and renders cells hypersensitive to IR [43]. The primary role of miRNA-182-5p is to mediate the dsDNA damage response, and its overexpression was found in a panel of human breast cancer patient samples, establishing its role as a potential oncomir in human breast cancer [43]. The miRNA-99 family reduces BRCA1 localization to sites of DNA damage, and introduction of these miRNAs into prostate and breast cancer cells reduces the rate and efficiency of repair by both HDR and NHEJ [43]. MiRNA-146a and miRNA-146b-5p expression is mostly high in some mammary tumor cell lines of the basal type with concomitant low levels of BRCA1 and in triplenegative breast cancers (TNBCs). MiRNA-146a and miRNA-146b-5p negatively regulate BRCA1 expression, and this down-regulation occurs with an increased proliferation and a reduced homologous recombination rate [29]. These results suggest that miRNA-146a and miRNA-146b-5p may play an important role in breast tumorigenesis by silencing BRCA1. Furthermore, differential regulation of BRCA1 in cancer cells with different genotypes appears to be associated with the BRCA1-interacting miRNA-638 [52]. Effectors of cell cycle checkpoints and apoptosis The ATM/ATR-CHEK2/CHEK1-TP53/MDM2-P21 pathway is the major one that controls the DNA damage-induced G1/S checkpoint and triggers G1 arrest. ATM/CHEK2 and ATR/CHEK1 are activated by DSBs and SSBs, respectively, 5.4

8

to phosphorylate and stabilize TP53 that in turn induce P21 expression. P21 inhibits the activity of CCNE/CDK2 and CCND/CDK4/6 complexes, leading to G1 arrest. The G1/ S cell cycle transition is controlled by a TP53-independent pathway, in which activated CHEK1/CHEK2 cause inactivation of CDC25A phosphatase by nuclear exclusion and ubiquitin-mediated proteolytic degradation, and consequently inhibits the CCNE/CDK2 and CCNA/CDK2 complexes. This TP53-independent pathway delays the G1/S transition only for a few hours, while the TP53-dependent checkpoint pathway prolongs G1 arrest. Two parallel signaling pathways mediate the DNA damage-induced intra-S checkpoint: CHEK1/CHEK2-CDC25A-CCNE(A)/CDK2 and ATM-NBS1-SMC1. In the first pathway, CDC25A degradation leads to the inhibition of CCNE/CDK2 and CCNA/CDK2 and delays replication by blocking the loading of CDC45 onto chromatin, which is required for the recruitment of DNA polymerase-a. As a consequence, this pathway extends the DNA replication time, allowing DNA repair to take place. In the second pathway, the ATM-mediated phosphorylation of NBS1 allows the recruitment of SMC1, which is in turn phosphorylated by ATM, leading to regulation of S-phase checkpoint. However, the precise mechanism is still not clear. DNA-damage-induced G2/M checkpoints are controlled by both TP53-dependent and TP53-independent pathways, which target the CCNB/CDK1 complex, leading to the promotion of mitosis. In the TP53-dependent pathway, ATM/CHEK2 or ATR/CHEK1 phosphorylate and stabilize TP53. TP53 induces P21 to suppress the activity of CCNB/CDK1, leading to late G2 arrest. In the TP53-independent pathway, CHEK1/CHEK2 phosphorylate and inhibit the three members of CDC25 family (CDC25A-C) and inhibit the activity of polo-like kinase 1 (PLK1), which is known to activate CDC25C. Furthermore, CHEK1/ CHEK2 can phosphorylate and upregulate the activity of WEE1 kinase, which catalyses the inhibitory phosphorylation of CDK1. All together, these CHEK1/CHEK2-mediate effects result in late G2 cell cycle arrest. Several miRNAs were found to regulate the expression of effector proteins involved in cell cycle checkpoints (Figure 2), including miRNA-449a/b, miRNA-21 and miRNA-16 targeting CDC25A, miRNA-195, miRNA-155 and miRNA-15 family targeting WEE1, miRNA-15 family suppressing CHEK1, miRNA-106b targeting P21 and miRNA-100 downregulating PLK1 [29,53,54]. If the DNA lesion is too extensive or cannot be repaired timely, apoptosis is initiated in order to eliminate the damaged cells, thereby preventing tumorigenic transformation. DSBs are thought to be crucial apoptosis-triggering lesions, and TP53 is a key factor involved in the DSB-induced apoptosis. In response to DSBs, ATM/CHEK2 and ATR/CHEK1 phosphorylate and stabilize TP53, resulting in transcriptional activation of pro-apoptotic factors, such as FAS, PUMA and BAX. To support TP53 actions, CHEK1/CHEK2 can also activate E2F1 and TP73, respectively, which in turn transcribe BAX,




PUMA and NOXA. While previous studies have concentrated on the stabilization and activation of TP53 proteins following DNA damage, recent investigations have tried to explore the potential relationship between TP53 and miRNAs. By searching conserved miRNA-binding sites in the 3¢ UTR of the TP53 gene, miRNA-125b was identified as a potential highly conserved TP53-targeting miRNA. Overexpression of miRNA-125b reduces endogenous TP53 level and suppresses apoptosis in human neuroblastoma cells and lung fibroblasts. In contrast, knockdown of miRNA-125b elevates the level of TP53 protein and induces apoptosis in human lung fibroblasts and in the zebrafish brain. These results suggest that miRNA-125b is an important negative regulator of TP53 and TP53-induced apoptosis during development and during the stress response [29,43]. Using a similar approach, miRNA-504 was identified as another TP53-targeting miRNA in human colon HCT116 TP53+/+, lung H460 and breast MCF7 cells [55]. MiRNA-504 inhibits TP53-mediated activities in human cells expressing wild-type TP53 and significantly increases the growth of human colorectal xenografts tumors in nude mice [55]. Furthermore, miRNA-375 was found to downregulate TP53 expression through an interaction with the 3¢ UTR region [56]. The expression of miRNA-375 desensitizes gastric cancer cells to IR and etoposide [56]. MDM2 is an E3 ubiquitin ligase and a known negative regulator of TP53. Following DNA damage, the TP53/MDM2 complex is disassociated, resulting in the rapid activation of TP53. MiRNA-605 was recently identified as a key cofactor in the TP53 regulatory network [57]. TP53 binds directly to the promoter region of the miRNA-605 gene, leading to its transcriptional activation. Transactivated miRNA-605 decreases the expression of MDM2 and thus indirectly enhances the transcriptional activity of TP53. These results reveal the existence of a TP53/miRNA-605/MDM2 positive feedback loop that ensures rapid accumulation of TP53 after DNA damage [57].

Role of the DDR-miRNAs in cancer tumorigenesis and progression

6.

Both DDR and miRNA pathways are involved in different stages of the carcinogenesis, from development to progression towards the gain of metastatic capacity. The aberrant expression of miRNAs and DDR components is a common feature of human malignancies [4,9,58]. Alterations of DDR pathways, as well as miRNA signaling, impair DNA repair, apoptosis and cell cycle mechanisms, thus influencing cancer biology. The importance of DDR alterations in tumorigenic process is demonstrated by a number of genetic disorders, such as ataxia-telangiectasia, Fanconi anemia, Rothmund-Thomson syndrome, Werner syndrome and xeroderma pigmentosum. Furthermore, hereditary non-polyposis colorectal cancers and a proportion of sporadic colorectal cancers are characterized by widespread alterations in the length of microsatellite sequences, known as microsatellite instability (MSI), due to a defective MMR pathway [14]. Similarly, a subset of both

sporadic and familial breast, ovarian and pancreatic cancers are associated with defects in HDR genes, such as BRCA1, BRCA2, PALB2, ATM and RAD51 [14]. The identification of alteration in DNA repair genes in cancer-prone human syndromes and in a subset of hereditary and sporadic cancers clearly indicates a causative association between DDR, genetic instability and cancer. Deregulation of the DDR commonly occurs in precancerous lesions and drives tumor development by increasing the spontaneous mutation rate [59]. Loss of genomic stability and accumulation of mutation in ‘caretaker’ genes may thus alter cell behavior, conferring a selective growth advantage and promoting tumor heterogeneity, cancer initiation and progression [59]. Genomic instability may also impact miRNA expression and processing. In particular, miRNA genes are frequently located at fragile sites, as well as in regions prone to amplification, deletion or common breakpoint regions [9]. Dysregulation of miRNAs can thus in turn contribute to the impairment of key pathways involved in tumorigenesis, directly promoting genomic instability due to their incontrovertible link with the DDR. For instance, miRNA-155- and miRNA-21-dependent downregulation of MSH2-MSH6 and MLH1-PMS2 results in a mutator phenotype characterized by a significant increase in mutation rates and MSI in colorectal cancers [42,60]. Moreover, miRNAs directly targeting DNA repair genes, such as BRCA1 (e.g., miRNA-181, miRNA-182 and miRNA-146) might be involved in establishing ‘BRCAness’ traits. There is enough evidence that link DNA repair genes with the onset of tumorigenesis, although a direct role of DDR in the metastatic process has only recently emerged. Cancer cells, particularly metastatic cells are characterized by genomic heterogeneity, which is a result of the presence of distinct cellular subclones originated by a progressive gaining of genetic aberrations. The accumulation of gene mutations and chromosomal instability has been demonstrated to increase tumor heterogeneity promoting tumor metastases. A recent study has demonstrated that only primary tumors having an altered expression of DNA repair-related genes had metastatic capabilities at specific organ-specific sites [61]. By gene expression profile analysis of breast cancers and matched brain metastasis, the authors found two specific DNA repair genes BARD1 and RAD51 directly involved in the metastatic process [62]. This data suggests that cancer cells with higher metastatic capabilities within primary tumors overexpress genes responsible for efficient DNA damage sensing and repair, ultimately resulting in genetically stable cellular clones able to colonize distant sites. These metastatic-competent cells may be identified as mesenchymal-/stem-like cells. Indeed, specific properties of cancer stem cells (CSCs), including high capability of tumor formation and metastatic invasion, are known to be correlated with an enhanced protection of genome stability due to a strong activation of the DNA damage sensor and repair machinery [63]. Although a unifying hypothesis regarding the connection between the DDR and the metastatic spread is still lacking, several miRNAs with known functions in the DDR


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Table 1. MicroRNAs and their respective DNA damage response targets involved in response to chemotherapeutic agents.


miRNA

Targets

Cancer

miRNA-138

ERCC1, H2AX

miRNA-182

BRCA1, CHEK2

miRNA-181a/b

BRCA1, ATM, BCL2

miRNA-155

WEE1, RAD51

miRNA-15 miRNA-96

CHEK1, WEE1 RAD51, REV1

miRNA-107 miRNA-221/222

RAD51 RAD51

miRNA-25/32

REV3L

Burkitt’s lymphoma cell, lung cancer cells

miRNA-125b miRNA-34a

TP53, BAK1 SIRT1, BCL2

miRNA-21

MSH2, MSH6

miRNA-28 miRNA-146 miRNA-203

BRCA1 BRCA1 ATM

Breast cancer cells Prostate cancer cells, breast cancer cells Colon cancer cells, glioblastoma cells, breast cancer cells, lung adenocarcinoma cells, pancreatic adenocarcinoma, CLL Breast cancer cells Breast cancer cells Colorectal cancer cell lines and tissues

NSCLC cells, osteosarcoma cells Embryonic kidney cells, breast cancer cells, cervix adenocarcinoma cells Breast cancer cells, CLL

Epidermoid carcinoma cells, triple-negative breast cancer, CLL Epidermoid carcinoma cells Breast cancer cells, osteosarcoma cells, ovarian cancer cells and tissue, cervix adenocarcinoma cells Breast cancer cells Breast cancer cells, CLL

Effects Chemosensitivity

Drugs

Ref.

Platinum agents, camptothecin PARP inhibitors

[40,67]

PARP inhibitors, platinum agents, fludarabine Platinum agents, taxanes, anthracyclines,

[45,94]

Chemoresistence Chemosensitivity

Platinum agents Platinum agents, PARP inhibitors

[53] [68]

Chemosensitivity Chemosensitivity, chemoresistance Chemosensitivity

PARP inhibitors PARP inhibitors, fludarabine Etoposide, camptothecin, PARP inhibitors Taxanes Taxanes

[69] [69,85]

Chemoresistence, chemosensitivity

5-FU, anthracyclines, taxanes, platinum agents, gemcitabine, fludarabine, cyclophosphamide, rituximab

[60,75-78,83,85]

Chemoresistence Chemoresistence Chemoresistence

Anthracyclines Platinum agents Platinum agents

[79] [81] [82]

Chemosensitivity

Chemosensitivity

Chemoresistence, chemosensitivity

Chemoresistence Chemoresistence

[43]

[53,88-91]

[71]

[72] [73,74]

PARP inhibitors mediate their antitumor effects by blocking repair of DNA single-strand breaks and enhancing the efficacy of DNA-damaging chemotherapeutic agents. 5-FU: 5-fluorouracil; CLL: Chronic lymphoblastic leukemia; miRNA: MicroRNA; PARP: Poly (ADP-ribose) polymerase.

pathways clearly play a role also in the regulation of CSC functions, epithelial-to-mesenchymal transition (EMT) and metastatic processes. MiRNA-21, miRNA-34, miRNA106b-25, miRNA-145, miRNA-200c and let-7 family have been directly involved in the regulation EMT and stem cell biology, through the targeting of key genes, such as SLUG, SNAIL, NOTCH, ZEB1/2 and CDH2 [64]. For example, miRNA-34 was found to repress both DDR genes, including BCL2, CCND1, CCNE2, CDK4 and CDK6, as well as NANOG and SOX2 pluripotency genes, globally acting as tumor and metastasis suppressor [64]. Other DDR-related miRNAs have been generally identified as metastasissuppressors or metastasis-promoters, including miRNA-17-92 family, miRNA-24, miRNA-155, miRNA-221/222 and miRNA-373 [65]. Interestingly, miRNA-155 has been demonstrated to both decrease invasion and promote EMT and metastasis in breast cancer, showing a double-edged role in 10

the regulation of tumor progression, as well as of DDR pathways [65,66]. All together, these data suggest the need of a better understanding of the underlying mechanisms regulated by miRNAs-regulated DDR pathways in cancer initiation, progression and therapy response. 7.

miRNA-DDR and chemotherapy sensitivity

The complexity and heterogeneity of tumor biology could lead to variability in response to conventional chemotherapeutics. MiRNAs may have important roles in altering chemotherapeutic response, especially through the modulation of the DDR. The most important miRNAs and their target DDR genes involved in drug response are listed in Table 1. Several DDR-related miRNAs were found to be deregulated and to modulate chemotherapy sensitivity, by both inducing quiescence or enhancing DNA repair or increased




DNA damage tolerance in distinct cancer types, including breast cancer and NSCLC [12]. The upregulation of miRNA-138 increased the sensitivity of resistant cells to cisplatin and apoptosis. MiRNA-138 was shown to target the excision repair cross-complementation group 1 (ERCC1), suggesting that miRNA-138 could play an important role in the development of cisplatin resistance [67]. MiRNA-96, miRNA-107 and miRNA-221/222 were recently found to impair DSB repair by HR and to enhance cellular sensitivity to various DNA damaging agents, including cisplatin and a Poly (ADP-ribose) polymerase (PARP) inhibitor by repressing expression of RAD51 and REV1, suggesting that these miRNAs can be potentially therapeutic agents for improving the efficacy of conventional chemotherapy [68,69]. MiRNA-25/32 was found to downregulate REV3L [70], a component of DNA polymerase zeta that interacts with REV1 to promote translesion DNA synthesis and DSB repair. Cells lacking REV3L are hypersensitive to topoisomerase poisons, PARP inhibitors and cisplatin [70,71]. MiRNA-125b, a negative regulator of TP53 and TP53-induced apoptosis, is upregulated in taxol-resistant breast cancer cells, and its overexpression causes a significant inhibition of taxol-induced apoptosis and increased resistance to taxol [72]. MiRNA-34a overexpression was found to attenuate paclitaxel resistance of paclitaxel-resistant prostate cancer cells, suggesting that miRNA-34a and its downstream targets SIRT1 and BCL2 play important roles in the development of taxane resistance [73]. Increased miRNA-34a expression is also associated with acquired docetaxel resistance and changes in miRNA-34a expression were found to modulate response to docetaxel in breast cancer cells [74]. MiRNA-21 is linked to several human tumors including colorectal cancer, where it appears to regulate the expression of tumor suppressor genes. It has been demonstrated that miRNA-21 targets the MMR proteins, MSH2 and MSH6 [60]. The MMR system is involved in DNA damage sensing and repair and defects in MMR-related proteins have been correlated with lack of response to 5-fluorouracil (5-FU) adjuvant chemotherapy in clinical trials [60]. MiRNA-21 overexpression causes a reduction in 5-FU-induced damage arrest and apoptosis, suggesting that miRNA-21-mediated downregulation of MSH2 and MSH6 might be responsible for resistance to 5-FU [60]. Furthermore, the MSH2 and MSH6 genes are associated with pathological complete response to taxane/anthracycline-based regimens in ER-negative/HER2-negative breast tumors [58]. MiRNA-21 inhibition was found to increase the chemosensitivity to taxol in glioblastoma cells and breast cancer cells, suggesting that miRNA-21 inhibitor therapy combined with taxane-based chemotherapy might represent a potential novel therapeutic approach for the treatment of several malignancies [75,76]. Furthermore, a TGF-b/miRNA-21/MSH2 axis was found to contribute to resistance of breast cancer cells to DNAdamaging chemotherapy agents such as cisplatin and doxorubicin, but not docetaxel [77]. However, cisplatin was found to inhibit lung adenocarcinoma cells growth in vitro and

in vivo by downregulation of miRNA-21 expression and upregulation of MSH2 expression [78]. MiRNA-28 and miRNA-146 were shown to target BRCA1 and to be deregulated in breast cancer cell lines resistant to genotoxic agents, such as doxorubicin and cisplatin [79-81]. The overexpression of miRNA-182 may play a role in BRCA1 downregulation in sporadic breast cancers. Indeed, overexpression of miRNA-182 was found to reduce BRCA1 and CHEK2 proteins, impair HDR and render cells hypersensitive to IR and to PARP inhibitors in both cultured cells and in animal models [43]. High levels of miRNA-181a/b may dampen BRCA1 and ATM functions and the deregulated expression of miRNA-181a/b is associated with sensitivity of TNBC cells to the PARP inhibition and platinum-derived compounds [45]. Moreover, a significant reverse correlation between miRNA-203 and ATM expression was found to be involved in oxaliplatin resistance in colorectal cancer [82]. Overexpression of the kinases WEE1 and CHEK1 was found to occur in cisplatin-resistant cancer cells. MiRNA-15 family and miRNA-155 expression was found to be downregulated in cisplatin-resistant cells. The ectopic expression of the two miRNAs sensitizes the cells to cisplatin-induced apoptosis by targeting WEE1 and CHEK1 kinases. Despite kinase inhibitors against CHEK1 and WEE1 are already in clinical trials, these results suggest that the increase of miRNA levels with a mimic may be a potential strategy to sensitize cancer cells to chemotherapy and overcome resistance to kinase inhibitors [53].

DDR-related miRNAs as predictive biomarkers in the clinical setting

8.

As detailed above, in vitro and in vivo studies have identified significant DDR-related miRNAs whose expression patterns correlate with drug response, although these findings have yet to be fully translated into the current clinical practice. Translational studies within ~ 200 clinical trials (clinicaltrials.gov) are currently aiming to identify the potential role of different miRNAs as reliable predictive biomarkers of response to treatments. In fact, miRNAs are known to be characterized by a remarkable stability both in technically challenging samples, such as formalin-fixed paraffin-embedded specimens and body fluids, along with a high sensibility and tissue-specificity in their expression patterns. Hence, the detection of altered DDR-related miRNAs affecting therapy response and inducing chemoresistance may be helpful in determining the optimal and alternative treatments for cancer patients. The uncovering of this class of small molecules, as for other types of biomarkers, are more likely to impact the clinical practice, resulting in the selection of patients who are most likely going to benefit of specific treatments, thus avoiding unnecessary therapies with potential side effects. Among DDR-related miRNAs, miRNA-21 expression has been associated to the prognosis and therapeutic response in a number of human tumors. Particularly, in pancreatic ductal


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G. Bottai et al.

adenocarcinoma miRNA-21 is associated with the overall survival (OS) of patients treated with gemcitabine. Multivariate analysis confirmed that patients with high miRNA-21 expression had a significantly shorter OS both in the metastatic and adjuvant settings [83]. Other findings highlighted that plasma levels of miRNA-21 are able to successfully predict chemotherapy response in lung, oesophageal and prostate cancers [84]. A study by Ferracin et al. examined the patterns of miRNA expression in a cohort of 29 chronic lymphoblastic leukemia (CLL) patients receiving fludarabine. The tumors of those patients were either initially resistant or became resistant to fludarabine over time. This study identified a panel of miRNAs, which was able to discriminate patients with clinical response from those resistant. This panel included miRNA-21 and miRNA-222, which exhibited a significant overexpression in non-responder patients both before and after the treatment with fludarabine. In addition, the investigators demonstrated that the dysregulation of these miRNAs could be linked to many TP53-responsive genes, suggesting their potential involvement in the establishment of fludarabine resistance phenotype [85]. Importantly, inhibition of miRNA-21 and miRNA-222 by anti-miRNA oligonucleotides induces a significant increase in caspase activity in fludarabine-treated TP53-mutant cells, but whether or not the alteration in miRNA levels are dependent on TP53 activation is currently under investigation [85]. Another DDR- related miRNA, miRNA-125b, exhibits increased expression levels in sera from patients with a poor sensitivity to neoadjuvant chemotherapy both in breast cancer and NSCLC [86,87]. Recent data reported the prognostic role of four miRNAs, including two DDR-related miRNAs, such as miRNA-155 and miRNA-27a, in TNBC tissues. However, further validation studies are warranted for this subset of miRNAs particularly for miRNA-155, which has been also demonstrated to be expressed in inflammatory cells. Despite these considerations, at least miRNA-27a seems to predict the clinical outcome of TNBC patients treated with anthracycline- or taxane-anthracycline-based regimens [88]. A different study reported that the overexpression of miRNA-155 was associated with good prognosis in TNBC, and high miRNA-155 levels were associated with lower RAD51 expression and with better OS in patients treated also with radiotherapy [89,90]. However, in this context, the role of miRNA-155 in TNBC needs to be further defined. Conversely, a different study demonstrated that miRNA-155 upregulation is associated with late-stage, lymph node metastasis, TNBC and poor prognosis [91]. Furthermore, Kong et al. found high miRNA-155 levels in breast tumor tissues from patients who experienced recurrence under chemo- and radiotherapy, suggesting different roles for this miRNA in mediating chemotherapy response [92]. Additional studies proved a role of miRNA-155 as a marker of response to chemotherapy. The overexpression of this marker was found to be associated with poor response to therapy in CLL patients. The reduced expression levels of 12

miRNA-155 in the plasma were linked to better OS, even when CLL patients were stratified for response to therapy. In addition, both previously untreated and relapsing patients with lower expression of miRNA-155 were more likely to experience complete response to fludarabine, cyclophosphamide and rituximab chemo-immunotherapy compared to patients with higher miRNA-155 expression. This data supports that plasma levels of miRNA-155 may be used as predictive marker to identify CLL patients who are less likely to experience a complete response to chemotherapy [93]. Even more, in CLL, underexpression of miRNA-181a and miRNA-181b was found associated with shorter OS and treatment-free survival in affected patients. In this study, the fludarabine-sensitizing effects of miRNA-181a/b were associated with downregulation of the antiapoptotic proteins BCL2, MCL1 and XIAP, known to be associated with leukemic cell resistance to chemotherapeutic agents. In addition, miRNA-181a/b expression was demonstrated to correlate with TP53 status, which means that the apoptosis-inducing effects of miRNA-181a/b may require TP53 activation [94]. Another study from Ohyashiki et al. reported that the plasma levels of miRNA-92a were markedly decreased in patients with non-Hodgkin’s lymphoma, and partially restored after the complete remission in response to chemotherapy (rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP] or CHOP-like regimens). Interestingly, the levels of miRNA-92a decreased again in patients with relapsed disease, suggesting that serum levels of miRNA-92a could be a useful marker, both for evaluation of therapeutic efficiency and prediction of recurrence [95]. 9.

Conclusion

Several miRNAs are induced by DNA damage and in turn regulate DDR. Several core proteins in the DDR pathways are regulated by miRNAs. DDR-regulated miRNAs and miRNAs that target the DDR are involved in the initiation and progression of tumorigenesis and also modulate the sensitivity of cells to chemotherapeutic agents [19]. Defects in the DDR/ DNA repair pathways and deregulation of miRNAs are known hallmarks of many types of human cancer [96,97]. Altogether, the examples reported above underscore the potential to use DDR-miRNAs as biomarkers for predicting prognosis and particularly response to chemotherapy. These data on miRNAs as predictive markers of response to therapy need to be interpreted cautiously. MiRNAs may have a different role in different types of tumors. Moreover, these concepts highlight the urgent need to clarify the cell and tissue specificity, and the role for each deregulated miRNA identified. Furthermore, attention should be paid to the source of miRNA. Tissue and plasma miRNAs may have a complete different prognostic impact. Finally, to validate the role of this specific class of miRNAs in DDR/DNA repair pathways and to demonstrate their potential for targeted-therapies, further investigations are warranted. In conclusion, the



understanding of the cross-talk between miRNAs and the DDR/DNA repair pathways may provide new insight into the alterations of cellular processes, which occur after DNA damage, and may shed light on the mechanisms that lead to chemotherapy response, ultimately suggesting better approaches of treatment.


10.

Expert opinion

The available evidences suggest that DNA damage signaling participates in miRNA biogenesis by regulating both transcriptional and post-transcriptional mechanisms. However, further studies are required to understand the correlation between the DNA damaging signaling and the miRNA processing. While the majority of the studies examining miRNA regulation in response to DNA damage have focused on events that occur in the nucleus, it will be important to extend the investigations to understand the contribution of cytoplasmic regulation of miRNA biogenesis following DNA damage. Furthermore, it will be attractive to determine whether DNA damage signals can modulate the turnover, stabilization, modification and degradation of miRNAs. The activity and the localization of miRNA biogenesis machinery components within the cell might be post-translationally regulated. In fact, several distinct phosphorylation sites have been found on these processing proteins, and phosphorylation of Drosha seems to be required for its nuclear localization and pri-miRNAs processing [43]. It is likely that other components in the miRNAs biogenesis machinery may be direct or indirect targets of DNA damage pathways. Furthermore, the export of pre-miRNAs from the nucleus to the cytoplasm could be induced by DNA damage. It has been found that the expression level or binding activity of several exportin5-interacting proteins could be altered upon DNA damage [98]. It has also been recently shown that inactivating mutations of exportin-5 lead to pre-miRNAs accumulation in the nucleus and thus to reduction of miRNA processing, whereas the restoration of exportin-5 function reverses the impaired export of pre-miRNAs [99]. Additional studies are required to determine the role of DNA damage in the regulation of the nucleus-cytoplasm shuttling mediated by exportin-5. DDR defects are known to promote the development of different types of cancer characterized by genomic instability but can also induce sensitivity to chemotherapeutic drugs in cancer cells [19]. Chemotherapy is one of the main anticancer strategies. However, therapy resistance, which can be either

innate or acquired, represents a major barrier for the successful treatment of cancer [13]. Cellular resistance to chemotherapeutic agents can be achieved through a variety of molecular mechanisms, including the impairment of the DDR pathways involved in DNA repair, apoptosis and senescence [59]. Due to the heterogeneity in terms of chemotherapy-sensitivity among different cancer patients, additional efforts should be made to identify novel predictive markers of therapy response. Considering the important roles of DNA damage-responsive miRNAs and miRNAs that regulate DDR and DNA repair genes, understanding these responses and their regulation may help to identify and overcome underlying mechanisms of resistance and to guide the choice of ‘personalized’ therapy. Furthermore, miRNAs cross-talk with DDR components could be thus considered a promising direct target for enhancing the effects of standard chemotherapeutic agents. MiRNAbased therapy, in combination with conventional or targetedtherapies, may represents a potential option for the clinical management of drug-resistant cancers. Recent efforts are directed towards the development of novel therapeutic platforms that potentially deliver specific miRNA mimics or anti-miRNAs associated with new types of chemistry. Accordingly, miRNAs and miRNA-targeting oligonucleotides may become promising tools for cancer treatments. MiRNAs can be identified as potential predictors of response to chemotherapeutic treatments within clinical trials. Further efforts to answer remaining questions will lead to the development of novel diagnostic and therapeutic strategies for many human neoplastic conditions with DNA damage processing defects.

Acknowledgements The authors are grateful to Fabrizio D’Adda Di Fagagna for the critical reading of the manuscript.

Declaration of interest L Santarpia has received AIRC Grant support (AIRC Grant6251). GA Calin is supported as a Fellow at The University of Texas MD Anderson Research Trust, as a University of Texas System Regents Research Scholar, and has received a US Department of Defense Breast Cancer Idea Award. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


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Affiliation Giulia Bottai1, Barbara Pasculli2,3, George A Calin*4 & Libero Santarpia†1 †,* Authors for correspondence 1 IRCCS Clinical and Research Institute Humanitas, Experimental Therapeutics Unit, Via Manzoni 113 - 20089 Rozzano, Milan, Italy Tel: +39 02 8224 5173; Fax: +39 02 8224 5191; E-mail: [email protected]; E-mail: [email protected] 2 University of Bari, Biotechnology and Pharmacological Sciences, Department of Biosciences, Via Orabona 4, Bari 70125, Italy 3 The University of Texas MD Anderson Cancer Center, Division of Cancer Medicine, Department of Experimental Therapeutics, 1881 East Road - Houston, TX 77054, USA 4 The University of Texas, The Center for RNA Interference and Non-Coding RNAs, MD Anderson Cancer Center, Division of Cancer Medicine, Department of Experimental Therapeutics, So Campus Research Building 3, 1881 East Road, Houston, TX 77030, USA Tel: +1 713 792 5461; Fax: +1 713 745 4528; E-mail: [email protected]

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Opportunities for translation: targeting DNA repair pathways in pancreatic cancer.

Targeting DNA repair pathways for cancer treatment: what's new?

Signaling pathways in cartilage repair.

The role of DNA repair pathways in cisplatin resistant lung cancer.

Comprehensive analyses of DNA repair pathways, smoking and bladder cancer risk in Los Angeles and Shanghai.

Cancer-specific defects in DNA repair pathways as targets for personalized therapeutic approaches.

The Impact of DNA Repair Pathways in Cancer Biology and Therapy.

Targeting Endogenous Repair Pathways after AKI.

How are base excision DNA repair pathways deployed in vivo?

Functions of PARylation in DNA Damage Repair Pathways.

An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation.

DNA repair pathways in trypanosomatids: from DNA repair to drug resistance.

MicroRNA biogenesis pathways in cancer.

Molecular pathways in prostate cancer.

Signalling pathways in endometrial cancer.

Cumulative defects in DNA repair pathways drive the PARP inhibitor response in high-grade serous epithelial ovarian cancer cell lines.

New insights into tumor dormancy: Targeting DNA repair pathways.

Exposure to Engineered Nanomaterials: Impact on DNA Repair Pathways.

Activation of DNA damage repair pathways by murine polyomavirus.

Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients.

Pharmacogenetics of the DNA repair pathways in advanced non-small cell lung cancer patients treated with platinum-based chemotherapy.

Pathways of DNA repair in T4 phage. II. Sedimentation analysis of intracellular DNA in repair-defective mutants.

Coinhibitory Pathways in Immunotherapy for Cancer.

Molecular Pathways Controlling Autophagy in Pancreatic Cancer.