TARGETING DNA REPAIR MECHANISMS IN AGGRESIVE NEUROBLASTOMA

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TARGETING DNA REPAIR MECHANISMS IN AGGRESIVE NEUROBLASTOMA

Bachelor Thesis Project in Biomedicine 30 ECTS Spring term 2021 Student: Rafael Ruiz Alarcón a20rafru@student.his.se Supervisor: Simon Keane simon.keane@his.se Examiner: Homa Tajsharghi homa.tajsharghi@his.se University of Skövde School of Health Sciences Box 408, 541 28 Skövde Sweden

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Abstract

Neuroblastoma is a tumour derived from cells of the nervous system and is the most common solid tumour in childhood. MYCN amplified and 11q-deleted neuroblastoma, two high-risk neuroblastoma were investigated in this study. RAD51 gene family includes six central genes for the dsDNA breaks repair by homologous recombination, which has been reported as important in varying types of cancer.

The study aims to investigate if the dysregulation of this gene family could be involved in the unstable genome of 11q-deleted neuroblastoma, and to better understand the link between both high-risk tumours. The RAD51 family genes’ expression level was measured by RT-qPCR in samples of 11q- deleted and MYCN-amplified neuroblastoma that were treated with a UVC treatment and were recovered during varying hours. R2 database and DAVID were used to study the RAD51 family’s expression levels, associated event-free survivability, and altered pathways. RAD51 family is highly dysregulated in these tumours, four genes of six were found to be altered in high-risk neuroblastoma.

Four of six genes presented altered expression levels in 11q-loss, and three of six in the MYCN- amplified case after the UVC treatment. The event-free survival probability analysis shown that the levels of expressions associated with high-risk neuroblastoma coincide with those that represent a poor life expectancy. Altered pathways were different in each type of tumour. 11q-deletion neuroblastoma’s pathways were associated with the nervous system development, and MYCN- amplified was related to the immune system. This study suggests that 11q-loss neuroblastoma presents a greater RAD51 family dysregulation compared with MYCN-amplified one, which could explain why its genome is unstable.

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Popular scientific summary

Neuroblastoma is the most common solid tumour out of the cranium in children. This cancer is developed during the embryonal stages of the child, and it is a tumour from the autonomous nervous system. A wrong development of the neural-crest tissues – a direct precursor of the nervous system – can cause the appearance of these tumours in the adrenal glands, which are located over the kidney.

In contrast to many other cancers, few causative factors have been identified in NB. Neuroblastoma is characterized by a sporadic appearance, which makes it especially hard to study. This cancer-like disease has a 7-8% of presence in children and corresponds to the 15% of deaths for cancer in this age group. The survival rates for this disease have a wide range depending on the risk of cancer, between 90% and less than 50%. The high-risk neuroblastoma survival rate is between 40 and 50%, and they represent half of all neuroblastoma diagnosed patients. However, this ratio can be even lower if the patients do not respond properly to the treatment, being below 20%. These patients continue to have poor outcomes, because it is one of the most difficult and challenging cancers to treat yet, despite the wide range of treatments that exists. These challenges justify the special efforts that are being made to face this disease, which is not well-known yet and affects hardly many children each year.

RAD51 gene encodes a protein with the same name: RAD51. This protein has a central function in the repair of the double-stranded DNA, our genetic material. When this DNA is broken by different factors, like UV radiation, it must be repaired quickly and properly. Depending on the type of break, different proteins and processes will be used to repair it. The RAD51 mentioned before can fix the double- stranded breaks in the DNA by a technique named homologous recombination repair. However, this protein does not work alone, it needs the help of the other five members in its family named RAD51 paralogs. The whole RAD51 family co-work with RAD51 and together repair the DNA. This family has been studied in many other cancers, and it seems to be related to them. This study aims to add a new grain of sand to the knowledge of neuroblastoma, trying to evaluate the association of the RAD51 family genes to high-risk neuroblastoma. The idea is to be able to determine if alterations in the expression level of RAD51 family genes are responsible for the genetic instability of some types of this cancer, having always on the horizon possible ways to use this knowledge in future treatments.

In this study, the expression of the RAD51 family has been studied in two different types of high-risk neuroblastoma: MYCN amplification and 11q chromosome deletion. The cells were treated with UV rays to break their DNA and study the homologous recombination repair. The expression of the RAD51 family was measured, and then an online public database called R2 was used to compare our results with published ones on the database with these same chromosome alterations. RAD51 family has been altered differently in the two types of neuroblastoma studied. 11q-deleted samples presented a greater RAD51 alteration, they have a worse repair capacity. Neuroblastoma genetic changes are related to the poor survivability of the patients. The low survival rates are also associated with changes in cell processes which are basic for cell functionality. As can be expected for cancer cells, neuroblastoma cells changes are also related to the immune system and the nervous system development.

This investigation could help in the development of new and more efficient therapies in the future. It is just a new piece in all the puzzle of cancer, but this study is a novel approach to neuroblastoma research.

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Abbreviations

BER Base excision repair BIR Break-induced replication DSB Double strand break DSBR Double strand break repair EFS Event-free survival/survivability FA Fanconi anaemia

GC Gene conversion GO Gene ontology

HR Homologous recombination HRR Homologous recombination repair MMEJ Microhomology-mediated end joining MMR Mismatch repair

MNA MYCN amplification NB Neuroblastoma

NER Nucleotide excision repair NHEJ Non-homologous end joining

RT-qPCR Reverse transcription quantitative polymerase chain reaction SDSA Synthesis-dependent strand annealing

SSB Single strand break SSBR Single strand break repair UVC Ultraviolet C

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Introduction

Neuroblastoma is the most common extracranial solid tumour in paediatric patients (Sanmartín et al., 2017). It is an embryonal tumour of the autonomic nervous system found most frequently in adrenal glands, meaning that the cell of origin is thought to be a developing and incompletely committed precursor cell derived from neural-crest tissues (Maris, 2008). Neuroblastoma represents one of the most challenging cancer for treatment decisions because of its unusual biological behaviour, frequently presents spontaneous regressions, maturation to ganglioneuroma, and a progressive high resistance against different treatments (Berthold & Hero, 2000). NB is responsible for between 7 and 8% of all childhood malignant diseases and 15% of all cancer-related deaths in this age group. It is the most diagnosed cancer during infancy. The average age of diagnosed patients is 19 months, moreover, 90% of NB cases are patients under 5 years old (Mlakar et al., 2017). In recent years there have been special efforts to establish different subgroups of patients, to improve their diagnosis and treatment.

For years these subgroups have been defined by many different characteristics like age at diagnosis, biological features, or the stage of the disease. These classifications are used nowadays, but the most important one is the classification by the risk of the patients (Berthold & Hero, 2000; Maris, 2008). The four different categories that were proposed are very low risk, low risk, intermediate risk and high risk for pre-surgical patients. They are based on the 5-year event-free survival rates: >85%, >75 to ≤85%,

>50 to ≤75%, and <50% respectively (Maris, 2008). These categories consider different characteristics such as tumour stage, histologic category, tumour differentiation, and several mutations in important genes like MYCN (Maris, 2008). Risk classification is commonly used to decide which type of treatment the patient needs. Most NBs are treated with typical therapeutic approaches for cancer-related diseases, including surgery, cytotoxic chemotherapy, and external beam radiation therapy. The standard therapies commonly use anthracyclines, platinum compounds, etoposide, vincristine, and different alkylating agents (Berthold & Hero, 2000; Brodeur, 2003). The standard treatment for very low and low-risk patients is the surgery, but if the disease becomes more complicated, chemotherapy is necessary (Berthold & Hero, 2000; Brodeur, 2003).

Many genetic features of NBs, such as the ploidy status, oncogene amplification or allelic loss, have now been identified that correlate with clinical outcome. For instance, near-triploidy is associated with favourable outcome, whereas MYCN oncogene amplification or allelic loss at certain sites, such as chromosome 1p or 11q, are linked with more aggressive tumours and poor prognosis (Brodeur, 2003;

Villamón et al., 2013).

Schwab and colleagues identified an oncogene related to MYC, MYCN (located in distal 2p), that was amplified from NB cell lines. This mutation is present in 17-20% of NB cases and is correlated with advanced stages of disease, a poor prognosis and a rapid tumour progression in all types of patients (Brodeur, 2003; Villamón et al., 2013). The MYCN amplification or MNA frequency increases to around 50% for high-risk patients. MYCN is a proto-oncogene, member of the family of MYC proto-oncogenes.

This family of proteins are basic-helix-loop-helix-leucine zipper or bHLH-LZ transcription factors which mediate mitogen signalling by regulating the transcription of different important genes involved in many processes like metabolism, biosynthesis, cell cycle regulation, DNA repair or cytoskeleton (Carén et al., 2010; Southgate, Chen, Curtin, & Tweddle, 2020). All of these processes are important in cancer- related diseases such as NB. MYCN does not present an extended expression in different tissues in the

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organism, its expression is mainly localised in the development of the nervous system. An ectopic expression of this oncogene leads to cell proliferation and the activation of tumour suppressor protein p53, sensitizing to apoptosis. It has been demonstrated that an increase in MYC activity drives to more aggressive NB because these oncogenes lead to rapid and erroneous replication, which result in replication stress. MYCN amplification also transcriptionally upregulates many proteins involved in the DNA DSB repair, like some components of the MRE11-RAD50-NBLS1 or MRN complex and A-NHEJ (Southgate et al., 2020).

Another common mutation in NB is the chromosome 11q heterozygous deletion, which is present in 30-40% of the total NBs, and many high-risk ones. MNA and 11q loss rarely occur together. The smallest region of 11q loss includes genes like CADM1 and other ones involved in the DNA damage response (DDR): ATM, CHK1, MRE11 and H2AFX (Mlakar et al., 2017). All of them have been studied as candidates of drivers for NB tumorigenesis. The 11q deletion leads to a loss of DDR proteins, which results in a chromosome instability that allows the development of cancer. H2AFX resides in 11q23.2- q23.3, a region which is generally lost in this type of tumours, however, all these genes have one copy remaining on the non-deleted chromosome, so they still have function. H2AFX encodes a core histone H2A variant, which is randomly incorporated in nucleosomes. It seems that the loss of one of the copies of H2AFX can enhance susceptibility to cancer in absence of p53, but this is not altered or deleted in NB. 11q deletion is associated with patients with particularly aggressive relapse and decreased overall survival (Carén et al., 2010; Southgate et al., 2020). Furthermore, in 11q-deletion cases, 17q gain is very common too, but segmental loss of several other chromosomes is present as well (Carén et al., 2010).

When any type of damage occurs to the DNA in cells, they either undergo apoptosis or try to repair the error. DNA damage is repaired by different pathways specific to the type of damage sustained (Southgate et al., 2020). These pathways work together to ensure any damage to DNA is repaired with high fidelity to maintain genome integrity. DNA lesions affecting one strand, such as SSBs, base deamination, oxidation, methylation or loss are repaired by BER, SSBR, and NER. PARP1 and PARP2 signalling is required for efficient SSBR. During the replication several errors occur, they are known as mismatches, which are repaired by the MMR pathway. In this pathway, errors in the newly synthesized strand are removed and the DNA is resynthesized by the replication machinery. However, DNA damage can affect both DNA strand too, like DSBs, replication stress and interstrand cross-links (Ray &

Raghavan, 2020). DSBs are deleterious lesions that if left unrepaired can lead to cell death, while if miss-repaired can give rise to genomic instability, leading, for example, to tumorigenesis (Ahrabi et al., 2016). These damages are repaired by NHEJ, HRR, the FA pathway, and MMEJ. Among the DSBR pathways, HR utilises homology-directed donor sequences and does not cause errors, while NHEJ mostly deals with error-prone repair resulting in indels or missense mutations at the target site. NHEJ has a much higher efficiency than HR, but it causes more errors (Southgate et al., 2020). The pathway choice between HR and NHEJ is dictated by the complexity of the break sites, owing to end-resection and cell cycle phases (Ray & Raghavan, 2020). In absence of certain NHEJ factors, MMEJ pathway acts.

MMEJ is the least accurate pathway. Its activation depends on HRR being inactive (Ahrabi et al., 2016;

Sharma et al., 2015).

In recent years, HR has been deeply studied as a key pathway for repair severe DNA damage like DSBs.

The RAD51 gene family includes RAD51 and five RAD51-like genes: XRCC2 (7q36.1), XRCC3 (14q32.33), RAD51L1 (14q24.1), RAD51L2 (17q22), RAD51L3 (17q12). All of them have important roles in the HRR

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pathway. This pathway is schematically represented in Figure 1, based on the one showed in Sullivan

& Bernstein, 2018. The function of the protein recombinase RAD51 is totally central in HR, it catalyses strand transfer between a broken strand and its non-damaged homologue, this allows the re-synthesis of the damaged region. The family is highly conserved, particularly in the putative ATP-binding domains. The function of RAD51 is to displace replication protein A or RPA to assemble nucleoprotein filaments with the 3’ ssDNA ends during the central step of HR. This step is highly regulated to prevent undesired recombination. This is when RAD51 mediators like BRCA2 and RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and SWSAP1) become important, because RAD51 filament assembly is stimulated by them. The RAD51 nucleoprotein filaments form a displacement loop or D-loop by invading a homologous region. The heteroduplex formed is then resolved, and the way it is resolved determine which pathway occurs GC, SDSA, and RAD51-dependent BIR. While RAD51 has emerged as Figure 1. Homologous recombination repair scheme. The picture shows briefly the steps followed by the HRR mechanism to repair DSB. The different steps are enumerated and the implicated proteins are mentioned in the steps they act. The key proteins to understand this study are marked with colours: green for RPA, red for RAD51, orange for complex CX3, and blue for complex BCDX2. The figure is based on the one presented by Sullivan &

Bernstein, 2018. Created with BioRender.com.

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a key player in replication through fork reversal, protection, and restart, the role of the paralogs in regulating RAD51 in these contexts awaits elucidation. Finally, deregulation of RAD51 and its gene family is associated with cancer and FA. Given the close association of RAD51 deregulation with cancer predisposition, understanding the underlying mechanisms of RAD51 gene family structure and function is critical for designing new cancer therapeutics and mechanistic insights into human disease (Bonilla, Hengel, Grundy, & Bernstein, 2020; Thacker, 2005; Xu et al., 2019).

RAD51 paralogs were discovered forming two different major complexes: BCDX2 and CX3. BCDX2 is formed by proteins RAD51B, RAD51C, RAD51D and XRCC2, while proteins RAD51C and XRCC3 are included in the complex. These complexes, as paralogs, play a central role in preserving genomic integrity by HRR pathway. Amongst all the RAD51 paralogs, RAD51C is thought to be the most important one because it plays a role in many cellular processes and is the only paralog found in both complexes. RAD51C seems to accumulate in break zones, as response to ionising radiation. RAD51 and RAD51C co-localize in these zones, and some studies have suggested that RAD51C has a main role in the recruitment of RAD51 paralogs. Those evidence are controversial and non-well-studied yet.

However, RAD51C functions do not finish there, because this paralog is the link between the DNA damage response and the HRR. RAD51C also plays a role in the cell cycle progression, being key for an efficient checkpoint signalling. This means that a proper HRR and a correct cell cycle progression are coordinated. Nevertheless, the exact mechanisms of action of these paralogs are still unknown (Bonilla et al., 2020; Suwaki, Klare, & Tarsounas, 2011).

RAD51 as a key protein of DNA HRR has been demonstrated to be really important in cancer diseases.

Higher expression levels of RAD51 are present in most tumours than in other tissues. These levels of RAD51 expression are associated with enhanced resistance to DNA damage induced by chemicals.

RAD51 expression is higher in high-risk NB than in low-risk NB (Xu et al., 2019). Genome sequencing studies have revealed that this enhanced expression is due to transcriptional and/or epigenetical increased (Xu et al., 2019). However, there are no studies that propose any type of association between RAD51 expression and both types of aggressive NB, MNA and 11q loss, in particular, and this fact may explain the differences between aggressive and standard NB.

The hypothesis is that 11q-deleted NB present a higher dysregulation of the RAD51 family after the induction of the dsDNA breaks and the recovery. The purpose is to compare both types of high-risk NB to understand the role of the RAD51 family in these tumours and if the existing unstable genome in 11q-deleted NB is cause for the putative dysregulation.

Materials and methods

Two different cell lines were independently studied in this investigation: SKNAS (11q-deletedcell line ATCC SKNAS CRL-2137) and SKNBE (MNA cell line ATCC SKNBE CRL2271). The varying samples of these cell lines were subjected to a UVC treatment for 60 seconds. After the induction of DSB through the radiation, the samples were divided by the time of recovery after the mentioned treatment. Four different subgroups were analysed in the study then: a control group, and three different times of recovery. The hours of recovery assayed were 0, 4 and 24 hours of recovery after the UVC treatment.

Three biological replicates of each cell line and time of recovery were analysed. All the samples were provided as freeze cell pellets.

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RNA extractions

To extract the RNA of the cells, the kit used was the RNeasy mini kit from Qiagen (Cat No: 74106). The process was carried out following the manufacturer’s guide. After the extraction, the RNA concentration of the samples was measured by Nano spectrophotometry using a DS-11 spectrophotometer/fluorimeter from DeNovix.

cDNA synthesis

RNA samples were used to generate cDNA, to further analyse them in RT-qPCR. In this step, the High- capacity cDNA reverse transcription kit from Thermo Fisher (Cat. No: 4368814) was used. To synthesize the cDNA, a Master Mix was prepared. The reagents and the volumes are according to the manufacturer’s recommendation. The cDNA synthesis was performed in 96-well PCR plates using the MJ Research PTC-200 Gradient Thermal Cycler (Cat. No./ID: MJ-G200). The programme used to run the cDNA synthesis was regular for the High-capacity cDNA reverse transcription kit, as per the manufacturer’s instructions.

Reverse-Transcription Quantitative PCR

To analyse the expression levels of the desired genes in the different samples, RT-qPCR analyses were carried out. Six different TaqMan probes were used to identify all the six genes corresponding to RAD51 family: RAD51D (Hs00982175_m1), RAD51B (Hs01568763_m1), RAD51 (Hs00947967_m1), RAD51C (Hs00427442_m1), XRCC2 (Hs03044154_m1) and XRCC3 (Hs00193725_m1). Two reference or housekeeping genes were also analysed to normalize the other gene’s expression: GAPDH (Hs02758991) and GUSB (Hs99999904). In total, the expression of eight genes was analysed.

To prepare the samples for correct RT-qPCR analysis, they were pooled with TaqMan Fast Advanced Master Mix (Cat. No./ID: 4444557). The proportion of reagents used for RT-qPCR analyses was 1 µL of the sample, 8 µL of free-nuclease H2O, and 9 µL of TaqMan Fast Advanced Master Mix. The concentration of the cDNA samples was between 5.10 and 99.4 ng/µL, as per manufacturer’s instructions. A total of 2 µL of this reaction mix were loaded per well in the microplates. Three technical replicates per sample with the same time of recovery were performed. When the plates were completely loaded, they were analysed using a Thermo Scientific PikoReal® 96 Real-Time PCR System.

After the analysis, the obtained data were treated using the Thermo Scientific PikoReal® Software 2.2.

These obtained expression data were all in a threshold of ±0.5 Cq for the three technical replicates assayed per each biological replicate of each time of recovery. Expression data were normalized against the mean expression of the housekeeping genes. These expressions were compared with the mean expression of the control samples per each analysed gene.

Gene expression analyses

In this study, the database R2 (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi) has been used to compare the expression levels of the genes in the RAD51 family in the two different chromosomal alterations investigated in high-risk NB. Different tools available in R2 were used to perform different analysis. The first tool used to compare the RAD51 family expression levels in patients with the different chromosome alterations have been compared with non-amplified-MYCN and non-deleted- 11q NB. Another R2 tool was used to create different Kaplan Meier graphs, comparing EFS probabilities

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against the months followed up depending on the expression of the RAD51 family genes. The last application utilized in R2 was which find differential expression between groups. It was used to generate two different gene list with enriched genes in both chromosome alterations studied. The dataset used for it was SEQC (gse49710).

. Differential gene expression was determined for MNA and 11q-deleted tumours using the R2 database. The samples were stratified based on MYCN status to investigate the differential expression in MNA and non-amplified tumours. To study how they are expressed in 11q deletion cases, samples without MNA were layered by high risk. A total of twelve boxplots were generated, six per each chromosome alteration investigated. The p-values of the associations were displayed by the database.

Kaplan Meier’s graphs were generated in R2 stratified by gene expression. The six genes were studied independently, one per Kaplan Meier plot, NB cases were divided by expression in two curves: low and high expression. The cut off mode used was the scan. The relation between the gene expression and EFS over time is given by the p-value and the Bonferroni correction value, which are shown in each graph.

To identify differentially expressed genes between 11q-deleted and MYCN-amplified NB the previously described dataset; SEQC was selected from the R2 platform. Differentially expressed genes for MYCN- amplified and 11q-deleted tumours were identified and ranked based on p-value, lowest to highest.

Two gene lists were generated in total, one for MNA patients and another one for 11q deletion cases, using the False Discovery Rate as multiple testing correction. The genes in both lists correspond to the enriched genes for the two chromosome alterations separately, comparing MNA and non-MNA tumours, and, 11q-deleted and non-11q-deleted cases.

DAVID analyses

Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) was used to relate the previously created gene lists with certain enriched GO terms and pathways. The gene lists include the enriched genes for MNA and 11q-deleted patients studied in R2 previously. The gene lists were uploaded and used to generate functional annotation charts as desired. The gene lists were used to determine different pathways and terms which are altered in these types of NB compare to low-risk tumours.

To only take into account relevant data and because DAVID only accepts until 6000 terms, the gene lists were not uploaded completely. MYCN amplified NB genes in the lists were selected by their p- value, using only those which had a p-value of less than 10^-2. However, in the gene list of NB with 11q deletion all the genes showed a p-value in that threshold, so only the first 6000 were utilized, which is the maximum in DAVID analyses.

The settings used in DAVID to generate both functional annotation charts were modified from the default settings. Functional annotation was limited to Level 5 or higher for biological process (BP) and molecular function (BP) These terms are more specific than defaults ones, allowing more precise results. After completing the settings, the different charts were created and studied.

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Results

SKNAS and SKNBE cell lines respond differently to UVC treatment

A total of four out of six genes presented altered expression levels after the UVC treatment and the recovery in SKNAS cell line: RAD51D, RAD51B, RAD51C and XRCC3. SKNBE cell line shows only three genes of this family altered: RAD51B, XRCC2 and RAD51C.

The gene expression results are represented in Figure 2, ordered by formed complexes. Both cell lines were analysed independently, to compare them later. The data shown are the normalized mean expressions for each gene and time of recovery studied, expressed as relative values. Green lines correspond to the SKNAS cell line and blue ones represent the SKNBE cell line. The cell lines are referred in the graphs as AS and BE to ease legibility. T-tests were performed to determine if significant differences in expression exist between varying times of recovery for the same genes. Some of those comparisons show significant differences between the means analysed, and they are coloured in red.

When a sample presents significant differences with the other two samples for the same gene, that sample is marked, but not the other ones. All of the significant differences present a p-value < 0.05, which is the first level of significance.

SKNAS cell line presents four genes with differential expression: RAD51D (A), RAD51B (B), RAD51C (E) and XRCC3 (F). RAD51D is overexpressed (p-value: 0.049) between 0 and 4 hours of recovery, and the difference of the expression is 0.134. RAD51B, RAD51C and XRCC3 are all significantly underexpressed.

RAD51B is downexpressed (0.011) between 0 and 4 hours, with a difference of 0.182. RAD51C is underexpressed (0.023) between 4 and 24 hours of recovery, and the difference in the expression between the samples is 1.362. The gene XRCC3 is downexpressed (0.047) too, in this case, the expression varies 1.050 between 0 and 24 hours of recovery.

SKNBE cell line shows three genes with significant differences in their expression over time. The three cases are similar: the mean expression of the samples with 24 hours of recovery is significantly different from the samples of 0 and 4 hours of recovery independently. The expression between the samples with 0 and 4 hours of recovery are not significantly different. The genes affected in these cell lines are RAD51B, XRCC2 and RAD51C. The gene RAD51B is underexpressed after 24 hours of recovery compared with the other two samples measured, 0 and 4 hours (0.033 and 0.038 respectively). The sizes of the changes are 2.932 and 3.675 respectively. XRCC2 is underexpressed in the same way as RAD51B is, the differences are 1.922 and 1.461 (0.025 and 0.038). RAD51C expression at 24 hours is downexpressed respect to the other two samples too (0.049 and 0.012). The differences between them are 2.519 and 1.719 respectively.

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R2 shows differential expression of the RAD51 family in high risk NB subtypes

Through the database R2, Figure 3 and Figure 4 were created to compare how the RAD51 family is expressed in both types of high-risk NB, MNA and 11q deleted respectively, against the normal NB.

Figures were created in R2 using varying tracks and subset tracks to compare MNA NB against non- amplified one, and 11q-deleted NB against normal one. MYCN status were studied in R2 comparing MNA and non-amplified cases. 11q chromosome deletion was also study comparing patients with and without the deletion.

Figure 3 shows how MNA NB express differently RAD51 family comparing with non-amplified tumours.

This high-risk cancer has four significant differences from the normal disease. RAD51D (A) and XRCC3 (F) are downexpressed in MNA NB relative to normal NB; however, RAD51 (C) and XRCC2 (D) are overexpressed (all p-values < 0.001).

Figure 2. Changes in relative expression of studied genes over time. Each graph represents the trends for the expression of the genes over the three times investigated: 0, 4, and 24 hours of recovery. Each plot correspond to one studied gene ordered by complex formation: RAD51D (A), RAD51B (B), RAD51 (C), XRCC2 (D), RAD51C (E), and XRCC3 (F). Green lines correspond to the SKNAS cell line expression and blue ones to SKNBE cell line. Markers coloured in red indicate significant differences between both markers. In the case of only one marker is red, it refers to that expression is significantly different from the other two independently, but the other ones are not.

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11q-deleted NB, as shown in Figure 4, present another four genes with differential expression between deleted and non-deleted cases. The gene RAD51D (A) is underexpressed in 11q-deleted patients.

RAD51 (C), RAD51C (D) and XRCC2 (E) are all of them overexpressed (all p-values < 0.001 except XRCC2’s p-value < 0.01).

B

C D

E F A

***

Figure 3. Differences in the expression of RAD51 family according to MYCN status. Graphs are ordered by investigated genes and the complex they form: RAD51D (A), RAD51B (B), RAD51 (C), XRCC2 (D), RAD51C (E), and XRCC3 (F). On the top of each boxplot, the p-value is shown and they are significantly different if the link between boxplots is marked with asterisks. * corresponds to a p-value < 0.05; ** are a p-value < 0.01; and *** means a p- value < 0.001.

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RAD51 family expression is associated with poor survivability

The Appendix shows six Kaplan Meier graphs of how RAD51 gene family expression influences the survivability of the patients. Plots show the EFS associated with high and low levels of expression for the genes investigated. Red lines correspond to the low expression of the genes, and blue ones

A

F E

C

B

D

***

*** **

Figure 4. Differences in the expression of RAD51 family according to 11q chromosome status. 11q deletion was selected utilizing the track high_risk and the subset track mycn_noamp, so 11q deletion is represented as yes in the boxplots. Graphs are ordered by investigated genes and the complex they form: RAD51D (A), RAD51B (B), RAD51 (C), XRCC2 (D), RAD51C (E), and XRCC3 (F). On the top of each boxplot, the p-value is shown and they are significantly different if the link between boxplots is marked with asterisks. * corresponds to a p-value < 0.05; **

are a p-value < 0.01; and *** means a p-value < 0.001.

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represent the high expression. The figure also reflects the p-value and the Bonferroni correction value corresponding to the Kaplan Meier analysis.

RAD51B is the only gene in the family whose expression changes do not present a significant difference associated with the EFS. RAD51 high levels are related to a poorer prognosis (bonf p: 5.9e-13). RAD51D and XRCC3 low expression is associated with a bad prognosis (1.9e-07 and 5.5e-3). The high expression of RAD51C and XRCC2 is related to poor prognosis too (1.5e-04 and 5.3e-08). The case of RAD51C is especially remarkable because the EFS decrease very quickly concerning the other genes.

Each type of NB present distinct altered pathways

The Gene Set Enrichment Analysis was performed with DAVID utilizing two different enriched gene lists, one per each chromosomal alteration investigated. The enriched lists of genes by each chromosomal alterations were analysed to determine to which pathways and GO terms were related.

The top five pathways’ results displayed are shown in Table 1. It is presented the number of genes correlated with that pathways or terms and the percentage they reflected. The p-value calculated by DAVID is also presented in the last column.

11q deletion cases have a top-five pathway where the development of the nervous system stands out.

Splicing processes and phosphoproteins are terms that are also present in the top five of both chromosomal alterations lists. MNA patients also show intracellular signalling as an important mechanism in this type of diseases.

Out of the top five for each NB type, there are also other remarkable pathways related to cancer, NB and both subtypes of it. 11q-deleted tumours have relevant connections with the cell cycle (p-value:

4.6e-15), neuron differentiation and development (1.1e-12 and 1.2e-12), neurogenesis (1.5e-15) and regulation of GTPase activity (2.9e-11) for example. The obtained pathways in MNA patients are the T cell and leukocyte aggregation (1.8e-19 and 1.9e-19), lymphocyte activation (2.7e-17) and small GTPase mediated signal transduction (1.2e-14). These results are more connected with the immune system and its response.

Table 1 Gene Set Enrichment Analysis performed by DAVID for both cell lines. The table shows the top five enriched terms for 11q deletion and MNA cases and their p-values.

Cell line Enriched terms #correlated genes Percentage p-value

11q deletion

Alternative splicing 3337 58.4% 1.0e-64

Phosphoprotein 2680 46.9% 1.8e-57

Splice variant 2531 44.3% 4.8e-45

Polymorphism 3546 62.0% 2.4e-31

Nervous system development 794 13.9% 4.4e-20

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Discussion

According to R2, and as presented in Figure and Figure 4, the genes belonging to the RAD51 family are mostly differently expressed between the high-risk NB and the non-high-risk tumours, but the changes in the expression are not large between both subtypes. RAD51D is underexpressed in MNA and 11q deletion patients in a similar way and size. RAD51B does not present significant differences in any of the investigated cases. RAD51 and XRCC2 are overexpressed in MNA and 11q-deleted NB, with a little bigger augment in MNA patients in concrete respect to 11q-deleted ones.

On the other hand, complex CX3 is differently expressed in the subtypes of NB too. XRCC3 is underexpressed in MNA patients, but it is not affected in 11q deletion cases. However, the gene RAD51C is not affected in MNA cases but is overexpressed for 11q-deleted NB sufferers.

The complex BCDX2 has not being affected in a different way between MNA cases and 11q deletion ones. However, the complex CX3 has suffered different changes depending on the type of the high- risk NB. Complex CX3 may be overexpressed in 11q deletion cases because RAD51C is overexpressed, and it could be downexpressed in MNA patients due to the diminution of expression in the gene XRCC3.

A total of four out of six genes present a varying expression in these types of NB. MNA tumours show two non-changing, two overexpressed, and two underexpressed genes. 11q-deleted patients manifest two non-changing, three overexpressed, and only one underexpressed gene. The total genes affected are the same for both subtypes, so it is difficult to be sure that one of the two subtypes has the HR more altered than the other one. It is clear that the HR pathway is concerned in these types of high- risk NB, but it has not been much investigated how they have altered and the magnitude of the changes previously.

The RAD51 family has been evaluated due to its importance in other cancer diseases, with the intention of elucidating its role in NB cases compared to those other cancers. However, there is a gap in the knowledge to the importance of the RAD51 family in NB. RAD51 is commonly overexpressed in many types of cancer such as pancreatic or breast cancer, being generally a marker for bad prognosis and advances stages of disease (Liu et al., 2017; Yu et al., 2017; Zhang, Ma, Yao, Li, & Ren, 2019). High levels of expression and presence of RAD51 even produce resistance for certain types of therapies against these tumours (Jia et al., 2019; Liu et al., 2017). The RAD51 levels are also high in NB cases, indicating also poor survival, advanced stages of the disease, larger tumour size, more possibilities of metastasis,

MNA

Phosphoprotein 1774 49% 1.2e-48

Alternative splicing 2151 59.5% 3.3e-44

Intracellular signal transduction 666 18.4% 2.2e-23

Protein transport 498 13.8% 2.4e-23

Cytoplasm 1037 28.7% 4.5e-23

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and poor tissue differentiation. The RAD51 overexpression leads the cells to promote the cell cycle transition G0/G1 phase, increasing probably the resistance to some anticancer drugs (Hu, Weng, Xia,

& Zha, 2020). Both subtypes studied, 11q deletion and MNA, show this gene overexpressed too, as might be expected from high-risk NB according to mentioned published literature.

The expression of all the other genes in the RAD51 family have barely been investigated, and only a few publications mention them. No published literature has been found concerning RAD51B, RAD51D and XRCC2 in NB. The gene RAD51C has been found in a publication about the role of PALB2 (partner and localizer of BRCA2), which interacts with RAD51, RAD51C, and XRCC3 during the HR (Hanenberg &

Andreassen, 2018). XRCC3 has been found in other publications about NB survivors and their risk to suffer a second disease of this type. XRCC3 was one of the candidate genes studied as putative related to the development of those second malignant neoplasm. It was determined that a significant association exists (Applebaum et al., 2017). This gene was also investigated in a study about the influence of the substance PCB153 in the DDR genes. However, no one of these studies displays a general view of how these genes are represented and act in NB (Gao et al., 2009).

Nevertheless, the HR is not only altered in standard conditions for NB, it is also altered in high-risk NB overtime after the UVC treatment. The differential changes in the expression between the varying hours of recovery investigated may elucidate a distinctive complex formation in the subtypes of NB.

One of the most relevant variations is the relative diminution of the RAD51C expression (Figure 1.E), which occurs in both subtypes of studied NB. RAD51C has been recently reported as the main initiator of HRR complex formation. RAD51C is part of the two complexes formed: BCDX2 and CX3, at the same time. RAD51C seems to adhere at the break zone, following the induction of DNA damage. This step is the first of all those required to repair properly the DNA in these cases. The correct functioning of the DNA repair mechanisms is key to preserve the chromosomes’ stability. Some investigations even relate how RAD51C could be a tumour suppressor gene. RAD51C role is still controversial due to different contrary studies, but it is not possible to refuse the importance of this gene and the corresponding protein in the HRR mechanism (Suwaki et al., 2011).

Lab results show a clear underexpression of RAD51C after 24 hours of recovery (Fig 1.E). SKNAS cell line presents the significant difference between 4 and 24 hours, while SKNBE exhibits it between 0 and 24, and 4 and 24 hours. The result is the same: a decrease in the expression of RAD51C after the first 24 hours. However, the size of the change is not the same in both cell lines, because SKNAS presents a difference of 1.362, and SKNBE ones are 2.519 and 1.719 respectively. SKNBE cell line has suffered a further decrease than SKNAS cell line, with a relative difference of 0.357. The general decrease of RAD51C can prevent the formation of the complexes involved in the HRR, deteriorating the stability of the chromosomes and, probably, reducing the life expectancy.

To terminate with the CX3 complex, the gene XRCC3 is downexpressed in SKNAS after 24 hours of recovery compared with the initial moment or time 0 (FIG 1.F). This gene is not significantly altered in the SKNBE cell line, whose basal expression is higher than SKNAS’ one. The two genes implicated in the formation of the complex CX3 are downexpressed after 24 hours in the SKNAS cell line, so, apparently, this complex hardly can be formatted and be functional in these cells. SKNBE presents a standard expression of this gene, but its RAD51C expression levels are lower, and this could indicate that the complex will not form either.

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The complex BCDX2 also displays a significantly different expression for some of its genes, so this key part of the HRR could be also altered in NB patients. RAD51 gene is not significantly altered after the induction of the breaks, so its function seems to not be modified due to it (Fig 1.C). On the other hand, RAD51B presents a significant underexpression in both cell lines (Fig 1.B). SKNBE cell line results in a modified expression between 24 hours of recovery and the other two times independently; while SKNAS shows it between 0 and 4 hours of recovery, decreasing the expression faster than the cell line with MNA. In this case, the 11q deleted cells present a faster but fewer decrease in the expression.

The gene RAD51D is overexpressed in the SKNAS cell line at 4 hours of recovery compared with the initial time (Fig 1.A). However, even before this increase, the SKNAS cell line presented a higher expression of RAD51D compared with the SKNBE cell line. The last gene analysed, XRCC2, is underexpressed in the SKNBE cell line at 24 hours after the UVC treatment compared with 0 and 4 hours of recovery independently (Fig 1.D).

A final count of altered expressions per cell line after the treatment reveals which cell line presents more alteration in the HRR. SKNAS has a total of four significantly different expressions over time after the UVC treatment, while the SKNBE cell line only presents three changes in the RAD51 family genes.

11q-deleted NB seems to be more sensitive to the UVC treatment over time, presenting probably a worse capacity to repair DNA. Both cell lines’ results show an HRR altered over time, unable to repair the breaks produced by the UVC treatment even after many hours of recovery.

Different studies have reported the high frequency of breaks and immense chromosomal instability which are related to 11q. Those studies suggest as a putative explanation that this chromosome region may include genes related to DNA repair. ATM, H2AFX and MRE11A are commonly included in the deleted region, and they are related to DNA repair and DNA stability, so these genes were reported as important in the problems concerning 11q deletions (Sanmartín et al., 2017; Takagi et al., 2017). These examples support the idea that 11q-deleted NB presents more susceptibility than other types of NB because they have an altered capacity to react against this type of DNA breaks due to the dysregulation of genes involved in DNA repair mechanisms, such as the RAD51 family.

The mentioned changes in the RAD51 family expression influence the EFS of the patients with NB.

Based on Figure 4, it is possible to know which level of expression for these genes presents a better life expectancy. The idea is to compare the expression of the RAD51 family with the Kaplan Meier curves to understand which trends are more beneficial.

All the four different genes in the BCDX2 complex are similarly expressed in 11q-deleted and MNA NB.

A higher expression of the RAD51 gene is associated with a reduced EFS (Fig 4.A). This gene is overexpressed in both subtypes of high-risk NB studied, indicating a possible cause of the augmented risk in these tumours. RAD51B gene is not significant differently expressed in the subtypes, but this gene is neither significantly related to an altered survival rate (Fig. 4.B). RAD51D is underexpressed in both tumours and low levels of expression are associated with poorer survivability (Fig. 4.C). XRCC2 gene presents an underexpression in MNA and 11q-deleted NB, which is related to a lower EFS probability (Fig 4D).

The expression of the genes forming complex CX3 is also relevant to determine the survival rate of NB cases, but in this case, 11q-deleted and MNA patients present differences in the expression of the genes. RAD51C overexpression is connected with a decreased survivability, and this gene is significantly higher expressed in tumours presenting 11q deletion (Fig 4.E). MNA patients do not have

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altered the expression of this paralog. Finally, a low expression of the XRCC3 gene is linked to a worse prognosis, and this gene is significantly downexpressed in MNA cases, but it is not altered in 11q- deleted patients.

The severity of the expression changes in each type of NB is similar due to the complex BCDX2 genes expressions. All the genes involved in this complex are expressed in the way that results in a worse prognosis. However, complex CX3 does present differences between both tumours. 11q deleted NB displays RAD51C overexpression, and this alteration is shown in the Kaplan Meier curves as deeply severe. The patients with this overexpression present a poor prognosis, rapidly decreasing the EFS.

This gene expression change seems to be more relevant than the one that occurs in MNA patients, which suffer a XRCC3 underexpression, associated with a low EFS too. Nevertheless, this change is displayed in the plot as a less sudden decrease or a smoother curve, so apparently, it is an alteration not as substantive. This difference between 11q-deleted and MNA sufferers supports the hypothesis raised about the challenging problem of chromosome 11q deletion challenge. 11q deletion cases appear to be more severe than other types of NB, and these results suggest that RAD51C expression can be related to the poor EFS that accompanied these tumours. Limited published literature has been found relating the survivability and the expression of these genes. RAD51 is the only one that appears to be investigated previously, showing similar characteristics to the presented before in this study (Carén et al., 2010; Liu et al., 2017; Zha, Hu, Weng, & Xia, 2020; Zhang et al., 2019).

GSEA results show a clear tendency to the whole process of the nervous system development in 11q- deleted cases. The neurogenesis, the differentiation and the development of the new neurons are relevant enriched pathways that coincide with the nature of the tumour. MNA patients present as enriched pathways processes related to the immune system: T cell and leukocyte aggregation, and lymphocyte activation. The immune system is important in tumours to create a micro-environment favourable for its growth, and even conferring protection against certain treatments (Wang, Luo, Cao,

& Ma, 2020).

On the other hand, both subtypes of high-risk NB share the enrichment of GTPases, small GTPase mediated signal transduction in MNA and regulation of GTPase activity in 11q deletion. GTPases are very important proteins responsible for many processes related to the transduction of signals, regulation of cell differentiation and proliferation, and transport of proteins and vesicles. This extensive family has been reported as enriched and altered in some types of NB. The dysregulation of these proteins can lead to the impairment of neurogenesis. So, these miss-regulations may lead to non-proper neurogenesis, developing a possible tumour in some cases (Molenaar et al., 2012).

The size of the altered pathways is completely immense, and they include all types of processes. This long list influences the proper functionality of the cells, preventing them from completing basic life functions. The altered pathways are key to understand how the poor prognosis of these subtypes of NB is developed because the miss-function of those disturbed processes leads the NB to behave as it does.

This study holds certain strengths and weaknesses. Probably the weakest part of the tasks that have been done correspond to the lab work. The concentrations obtained from the RNA extractions were low, being even under the threshold of the machine used to quantify these values. This may have influenced subsequent lab work, probably modifying some results. Low concentrations were identified soon and lab workflow was altered in consequence. Protocol steps were eliminated, the time utilized

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per sample was reduced, and new reagents were used too, but concentrations remained low after the changes. These issues could have influenced the obtained results but in any case, different interesting results and conclusions have been able to be described. Curiously enough, probably the main strength of the project has been also the lab work, the qPCR analysis. These experiments have been repeated between two and four times depending on the gene and the sample to ensure that obtained results were consistent with each other. After getting the results, they were cure using quality control methods, so they are supposed to be relatively accurate.

In conclusion, the RAD51 family seems to be more dysregulated in 11q-deleted tumours than in MNA cases. Both types of NB show a high grade of disturbance in all these genes, which results in an altered HRR in high-risk tumours. Four out of the six genes investigated present a significantly different expression in the high-risk subtypes compared with the standard NB. The HRR is also altered after the UVC treatment and the recovery, but now the differences between the cell lines are clearer. SKNAS cell line shows four altered expressions in the studied genes, while SKNBE only presents three. These levels of expressions suggest that 11q-deleted tumours respond worse against the treatment than in MNA cases. The SKNAS cell line could present a higher dysregulation, which is a putative cause of the unstable genome of these patients. All the alterations mentioned translate into lower EFS in the high- risk NB because they present levels of expression associated with a poorer prognosis. 11q-deleted tumours, in particular, show a high expression of RAD51C, in contrast with the MNA cases, which is associated with really bad life expectancy. Finally, the low EFS is related to the pathways and cellular processes affected in this cancer-like disease. The pathways altered present a wide-ranging nature and they are divided between high-risk subtypes. 11q-deleted cases show alterations in different nervous system processes, while MNA patients display mainly immune system pathways as first results. The GTPase activity and regulation are also important for both tumours.

Aiming for future researches on this topic, it would be interesting to study the differences in the expression of the RAD51 family after the UVC treatment between high-risk and non-high-risk NB to compare them. To further investigate the subject it might be possible to analyse other cell lines with these chromosomal alterations, or cell lines treated with common therapeutics, or different genes related to the HRR or the resulting enriched pathways.

Ethical aspects and impact of the research on the society

Each year, approximately 1500 cases of NB occur in Europe and 700 in the USA and Canada, accounting for about 28% of all cancers diagnosed in European and North American infants. In contrast to many adult cancers, few causative factors have been identified. NB is characterized by a sporadic occurrence, which makes it especially challenging to study. The survival rates for this disease vary depending on the risk of cancer, between 90% and less than 50%. The high-risk NB survival rate is between 40 and 50%. However, this ratio can be lower if the patients do not respond properly to the treatment, being so below 20% (Heck, Ritz, Hung, Hashibe, & Boffetta, 2009; Whittle et al., 2017). The high-risk NB account for approximately half of all diagnosed patients with NB. Despite the wide range of treatments strategies for patients with this type of NB, this group continues to have poor outcomes and is one of the most challenging to treat yet. This information justifies the special efforts that are being made to face this cancer, which is not well-known yet and affects severely many children each year.

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The protocol of this study included the collection indirectly and manipulation directly of human cells or tissues. This is an ethical issue to bear in mind. The study basis is to improve the knowledge about aggressive NBs and their relationship with the DNA repair mechanisms because this link can help to elucidate how UVC treatments interact with NBs, resulting in a possible improvement of the treatments used. Therefore, the aim of this study results in trying to understand better this disease to improve treatment options for children who suffer this disease, but not only the treatments, but also reduce the side effects. The RAD51 family has been shown key in this disease, and it could be a putative pharmaceutical target in future therapies or medicaments. The differential complex formation between both subtypes of NB could be a potential target in the development of future treatments.

This is also important to treat each type of tumour differently and more precisely, improving the survivability of the patients. It would be also relevant to rethink the need of radiation therapies as they perhaps do not work properly. This study is the foundation to show that better treatment opportunities exist.

These goals can be tried to be achieved thanks to those patients who in an altruistic way donate their cells. Patients who suffered from NB and are undergoing non-experimental diagnostic involvements can decide to donate their cells for scientific use. Donated cells are used in investigations, mainly genetic ones, but the characteristics of the study that will be developed are still unknown for everyone.

The donation of NB cells is an incredibly beneficial way to help to understand NB genetic basis and to try or understand how new and old treatments work. However, there can be a problem with new NB investigations because researchers cannot always find enough patients to do complete clinical trials on. They can use compassionate use exemption in these cases so, using NB children as test subjects.

Referred to NB associated health issues, patients that survive aggressive NBs that survive are subject to significant lifelong morbidity that impacts the individuals’ quality of life. Problems such as scoliosis, weakness in the limbs, seizures, growth deficiency, hearing loss and blindness are commonly associated with this type of cancer. However, the treatments on young developing children can also lead to other issues related to learning difficulties, required special education as well as impaired social skills. The combination of all of these factors can result in a lower quality of life for those patients who survive NB.

Nowadays, the available treatments to patients with aggressive neuroblastoma are not successful presenting survival rates that are below 35%. As these tumours can undergo repair the current treatment strategies are highly inefficient, invasive and often result in significant morbidity and long- term disability (Whittle et al., 2017). Understanding and characterizing this cancer is key to improve the existing treatments. To increase the survival and reduce the morbidity of current low efficacy treatments the first step is to understand how NB affects DNA repair pathways, especially the HR, and try to direct treatments to it. The HR pathway is commonly affected by different types of cancer, not only by NB, so resolving the regulation of this DNA repair mechanism will be beneficial to many cancer patients, not just to children affected by NB.

In this case, as childhood cancer, children’s will have been followed, but like children cannot give consent so it is the parents that must do it. Names of the children are not known but some personal information normally is. However, data has been de-identified because they are unnecessary to perform this type of investigation, only the type of NB is known.

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Acknowledgment

I would like to thank my supervisor Simon Keane for all his support and the theoretical and practical knowledge he has taught me during these months of work. I would also like to thank my family and friends for being fundamental pillars in my life in the form of support and affection always. Finally, a special thanks to my friend Núria Lladós, who has shared countless hours with me during this time, discussing the work done and helping me in absolutely everything without hesitation throughout this hard course. She has been instrumental for this study to have been possible.

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Appendix

Figure A1. Kaplan Meier graphs for genes belonging RAD51 family. EFS followed up in moths is represented per each studied gene. The genes are ordered by the complex they formed: RAD51 (A), RAD51B (B), RAD51D (C), XRCC2 (D), RAD51C (E), and XRCC3 (F). Blue curves show the EFS associated to high levels of expression for those genes. Red lines correspond to low expressions. The p-value and Bonferroni correction value are represented in the bottom-right corner.

A B

C

E F

D

TARGETING DNA REPAIR MECHANISMS IN AGGRESIVE NEUROBLASTOMA