DLG2 impairs dsDNA break repair and maintains genome integrity in neuroblastoma

doi:10.21203/rs.3.rs-495251/v1

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DLG2 impairs dsDNA break repair and maintains genome integrity in neuroblastoma

https://doi.org/10.21203/rs.3.rs-495251/v1

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published 01 Feb, 2022

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Background: In primary neuroblastoma, deletions on chromosome 11q are known to result in an increase in the total number of chromosomal breaks. Microhomology mediated-end joining (MMEJ) is an error-prone pathway for DNA double-strand break repair that is often upregulated in cancer. DLG2, a candidate tumor suppressor gene on chromosome 11q, has previously been implicated in DNA repair.

Methods: We evaluated an association between MMEJ gene expression and neuroblastoma patient outcome, risk categorization, and 11q status using publicly available microarray data from independent neuroblastoma patient datasets. Functional studies were conducted using comet assay and H2AX phosphorylation in neuroblastoma cell lines and in the fruit fly with UVC-induced DNA breaks.

Results: We show that the MMEJ genes PARP1 and FEN1 are over expressed in neuroblastoma and restoration of DLG2 impairs their gene and protein expression. When exposed to UVC radiation, cells with DLG2 over expression show less DNA fragmentation and induce apoptosis in a p53 S46 dependent manner. We could also confirm that DLG2 expression results in CHK1 phosphorylation consistent with previous reports of G2/M maintenance.

Conclusions: Taken together, we show that DLG2 expression increases p53 mediated apoptosis in response to genotoxicity, by maintaining S317 CHK1 phosphorylation and reducing the DNA replication machinery.

Cancer Biology

Molecular Biology

DLG2

DNA Damage

Neuroblastoma

Cancer

Neuroblastoma (NB) is a tumor arising from the embryonic neural crest, later presenting in the autonomous nervous system [1]. It is one of the most common forms of pediatric malignancies and has a disproportionately high mortality rate [2]. Clinical screening and eventual diagnosis of NB is difficult due to the vague appearance of symptoms which is compounded by the young age of the patients [3]. NB can be defined by risk group, both low and intermediate NB have good treatment prospects, whereas high risk tumors are difficult to treat with current protocols [4]. This difficulty to treat results in higher incidence of refractory NB [5] as well as higher mortality [6], indicating that new treatments are needed. High-risk NBs are highly aggressive, with specific genetic lesions, copy number variations (CNV), and structural chromosomal changes [7]. Common genetic features of NB include chromosome 1p deletion [8–10] and 17q gain [11, 12], whereas 11q deletion [7, 13, 14] and amplification of the proto-oncogene MYCN [15–19] account for approximately 30% and 20% of all NB cases, respectively. In NB, deletion of 11q has previously been shown to result in an increase in the number of chromosomes with DNA breaks as well as the total number of chromosomal breaks[7].

DNA strand breaks can be repaired using different mechanisms depending on the type of break. DNA repair of single-strand breaks will often be highly successful as the DNA template still remains. In contrast, double-strand break repair can be deleterious as there is no template remaining for use during DNA repair. There are two main mechanisms by which double-strand break repair occurs: 1) Homologous Repair (HR) is highly reliable and uses the sister chromatid as a template, but can only take place during the S and G2 cell cycle phases [20] and 2) non-homologous end joining (NHEJ) does not use a template and directly ligates the two ends of the DNA breaks, meaning that the repair mechanism can be active throughout the cell cycle, except during mitosis [21].

Two distinct mechanisms of NHEJ have been found to occur, namely canonical NHEJ (c-NHEJ) and alternative NHEJ (alt-NHEJ). c-NHEJ is a more accurate DNA repair system, which includes several genes, i.e. XRCC4, XRCC5, XRCC6, DCLRE1C, LIG4 and DNA-PKcs [21–23]. alt-NHEJ, also referred to as microhomology-mediated end joining (MMEJ), is highly erroneous as the homology sequences create overhangs which are subsequently removed, creating insertions, deletions and whole chromosomal losses [24, 25]. As MMEJ-mediated repair is rare in G1 but increases in S and G2 phases, it is active at the same time as HR. Although cells undergo MMEJ when they cannot successfully undergo HR [26], the repair process has been shown to be competitive. Genes involved in the MMEJ pathway include MRE11a (11q21), FEN1 (11q12), LIG3 (17q12), XRCC1 (19q13), NBS1 (8q21), and PARP1 (1p42) [24].

In NB, the 11q-deleted region spans a number of candidate tumor suppressor genes such as ATM, H2AX, CHK1 and MRE11a [6], all of which are involved in DNA damage response (DDR) [27]. Recently, 11q deleted NB was shown to have abnormally low expression of Discs Large Homologue 2 (DLG2; 11q14) [27, 28]. Low DLG2 expression has also been found in osteosarcoma [29] and ovarian cancer [30]. Previous results show that DLG2 can regulate the cell cycle as well as potentially regulating DNA damage repair [27]. All of the candidate tumor suppressors spanning the 11q region have been shown to lack the second hit as per the Knudson two-hit hypothesis [6]. It has, therefore, previously been suggested that there is a coalescence of haploinsufficiency within a single pathway[28]. However, this has not yet been investigated.

In this study, we investigate the difference in expression of genes associated with MMEJ in primary NB of various stages, and can show that some of these changes may be induced by loss of DLG2. Furthermore, we can show that DLG2 loss increases the presence of DNA double-strand breaks, and that restoration of DLG2 results in an increase in overall genomic stability.

Gene expression analysis

To compare gene expression between different NB patient subgroups (risk, patient survival, and chromosome 11q status), microarray data (centered log2 fold change) for primary NB datasets (i.e. Risk group GSE49710, Patient survival GSE16476, 11q GSE3960, and 11q GSE73517) were downloaded from the R2 platform (http://r2.amc.nl) as log2 centered values.

Cell lines and tissue culture

Human NB cell lines (SKNAS, SKNBE(2), and NB69) were obtained from the ATCC Cell Line Collection (ATCC). SKNAS and SKNBE(2) were maintained in RPMI 1640 culture medium supplemented with 10% FBS, 1% L-Glutamine, 1% HEPES solution, and 1% sodium pyruvate. NB69 was cultured in RPMI 1640 medium supplemented with 15% FBS, 1% L-Glutamine, 1% HEPES solution, and 1% sodium pyruvate. The cells were cultured in a humidified incubator at 37°C with 5% CO₂.

Plasmids, siRNAs, transfections and irradiation

The DLG2 isoform 7 (NM_001351274.2) over expression plasmid on a backbone of pcDNA3.1/C-(K)-DYK (OHuq102626D) vector was purchased from GenScript (Genscript Biotech Corporation). siRNA targeting DLG2 (cat. s4122) or Silencer™ Select Negative control No. 1 siRNA (cat. 4390843) were purchased from Ambion (Thermo Fisher Scientific). Cell lines were grown to 80% confluence and subsequently transfected with DLG2 plasmid, empty vector “mock” (pCMV6-AC-GFP), si-DLG2 or scrambled control “mock”. In brief, 500 ng plasmid DNA or 30 pmol siRNA was complexed with 12 µl Lipofectamine 2000 according to the manufacturer’s protocol (Thermo Fisher Scientific). To induce DNA damage, the cells were uncovered and subjected to UVC radiation (ESCO; CRF/UV-30A; 253.7 nm) for 30 seconds at 1200mm, immediately given fresh media, and allowed to recover for 2 hours or 24 hours prior to harvesting.

Comet assay

To determine DNA damage repair post UV exposure, comet assay was performed according to the manufacturer’s protocol (Abcam, Cat. ab238544). Aliquots of 10 000 cells were mixed with low melting point agarose and added to comet assay microscope slides. The slides were allowed to harden for 15 min, lysed (pH 10; 4°C) for 1 hour in pre-chilled lysis buffer, subsequently washed 3 times in alkaline buffer and transferred into alkaline buffer for electrophoresis. The slides were run for 30 min at 1V/cm, 300 mA, washed 3 times with H₂O, incubated for 5 min with ice cold 70% ethanol, allowed to air dry, and incubated with Vista green for visualization. Measurements and analysis were performed using Image J software version 1.52a. Data were normalized to the control transfection and expressed as a percentage of the control.

Quantitative PCR (qPCR) analysis

Total RNA from NB cell lines and flies was extracted using the RNeasy plus mini kit® (Qiagen) according to the manufacturer’s protocol. The RNA concentration was quantified by NanoDrop (NanoDrop Technologies) and 2 µg of RNA was reverse transcribed into double stranded cDNA on a T-professional Basic Gradient thermal cycler (Biometra) using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). cDNA corresponding to 20 ng of RNA for each qPCR reaction was used. qPCR was performed on a Pikoreal qPCR System (Thermo Fisher Scientific) in triplicate for TaqMan Human and Drosophila transcripts (Table 1), using TaqMan™ Gene Expression Master Mix (Applied Biosystems, Cat. 4318157). Quantitative gene expression data were normalized to the expression levels of the two human reference genes GAPDH and GUSB, or the fly reference gene Rpl32 (Table 1) using the Livak method [31, 32].

Table 1

**List of genes and Probe ID used for qPCR Investigation.**
Gene name	Probe ID
DLG2	Hs00265843_m1
BAX	Hs00180269
BCL2	Hs00608023
PARP1	Hs002422302
MRE11a	Hs00967437
FEN1	Hs00748727
XRCC1	Hs00959834
LIG3	Hs00242692
NBS1	Hs00159537
GAPDH	Hs02758991
GUSB	Hs99999904
dmDLG	Dm01799281_g1
PARP	Dm03419822
MRE11	Dm01817703
FEN1	Dm01821494
XRCC1	Dm795840
LIG3	DM02139557
Rpl32	Dm02151827

Apoptosis assay

Apoptosis levels were determined in the transfected NB cells to confirm the previous qPCR results by the Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega) by exciting at 485nm and reading at 520nm using a FLUOstar Omega multiplate reader (BMG Labtech), as per the manufacturer’s instructions. After the addition of the caspase assay, the plate was incubated for 65 min at room temperature. Apoptosis was calculated by subtracting the fluorescence for the average blank background and then normalizing against the mean of the control cells.

Fly strains and crosses

Commercially available control white (w1118) flies (Drosophila melanogaster) and UAS-RNAi-dlg1 flies were crossed with da-GAL4 driver strain to silence gene expression. All strains were obtained from the Bloomington Drosophila Stock Center (Bloomington). Twenty female da-GAL4 flies were crossed with 10 male UAS-transgenic flies or control flies and the progeny incubated at 25 °C on standard fly media. 10 flies per cross were subsequently harvested for subsequent RNA and DNA preparation. To determine the effect of UVC irradiation the crosses were subject to 30 Seconds UVC and 3 hours recovery on regular substrate.

Nanopore libraries and Sequencing

To prepare ONT libraries D. melanogaster gDNA was extracted with the Blood & Cell Culture DNA Kit (13323, Qiagen) using the manufacturer´s tissue protocol. Libraries were prepared with 1 µg gDNA using the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies) combined with native barcoding expansion 1–12 PCR free EXP-NBD104 (Oxford Nanopore Technologies) according to the manufacturer’s protocol. A single 24 h sequencing run for D. melanogaster were performed with an R9.4.1MinION flow cell.

Sequence alignment and quantification of DNA integrity

To quantify DNA breakage, a strategy of global alignment between the full length of the sequenced reads and the corresponding reference genome region, where the read best aligned was chosen. Initial alignment was performed with BLASTn version 2.9.0+. A custom BLAST reference database was created from FlyBase D. melonagaster version 6.38 (FB2021_02, released April 13, 2021) [33], filtered to only contain chromosomes. All reads passing quality control were subsequently BLASTed to this database using a gap opening and extension penalty of 1 and an e value cutoff of 1e-10. Only hits that uniquely mapped to the reference genome were considered for further downstream alignment. Global alignment was performed using the needle function from the Biopython module EMBOSS [34] on the full length of the read and the corresponding region of the same length in the reference genome. Every mismatch, insertion and deletion were counted in these alignments. To quantify fused sequences, previously discarded reads were counted that fit the following criteria: the read has exactly two BLAST hits and there is at least 10kb between the two hits to account for larger deletions in the sequenced DNA.

Western blot

Total protein was extracted from transfected cells in 24 well plates (1 × 10⁵ cells/well), by aspirating the media and incubating on ice for 5 min then adding ice cold RIPA buffer (Thermo Fisher Scientific, Cat. 89900). Western blot analysis was performed using a Mini-PROTEAN® TGX™ 8–20% gradient gel (Bio-Rad Laboratories), and protein blotted onto LF-PVDF membrane (8 min, 25 V and 2.5A) using a Trans-Blot® Turbo™ Transfer System (Bio-Rad Laboratories). Blots were subsequently blocked for 1 h in 5% milk in Tris buffered saline (TBS) (150 mM NaCl in 10 mM Tris–HCL, pH 7.4) buffer supplemented with 0.1% Tween-20, as per the manufacturer’s recommendations. Blots were probed overnight at 4°C with antibodies diluted in PBST (0.1% Tween-20 in phosphate buffered solution (PBS)). Primary antibodies for DLG2 (1:500, Cell Signaling Technology, Cat. D4Z4D), PARP1 (0.5µg/ml, DSHB Hybridoma, Cat. AFFN-PARP1-17B10 was deposited to the DSHB by EU Program Affinomics), FEN1 (1:1000, SCBT, Cat. sc-28355), PCNA (1:1000, SCBT, Cat. sc-56), H2AX (1:1000, SCBT, Cat. sc-517336), γH2AX (1:1000, SCBT, Cat. sc-517348), P53 (1:1000, SCBT, Cat. sc-126), S46 P53 (1:1000, SCBT, Cat. sc-377561), BAX (1:1000, SCBT, Cat. sc-20067), BCL2 (1:1000, SCBT, Cat. sc-509), CHK1 (1:1000, SCBT, Cat. sc-8408), S317 CHK1 (1:1000, CST, Cat. D12H3), CHK2 (1:1000, SCBT, Cat. sc-5278), T68 CHK2 (1:1000, CST, Cat. C13C1), ATM (1:1000, SCBT, Cat. sc-135663), DNA-PKcs (1:1000, CST, Cat. E6U3A) and GAPDH (1:2500, Bio-Rad, Cat. 12004168). The secondary antibodies used for detection were Starbright B520 goat anti-Rabbit (1:5000, Bio-Rad, Cat. 12005870), Starbright B700 goat anti-mouse (1:5000, Bio-Rad, Cat. 12004159), both of which were diluted in PBST 0.1%. All wash stages were 3 × 10 min in TBST 0.1%. Secondary antibodies were incubated for 1 hour at room temperature. Image detection was performed on ChemiDoc MP (Bio-Rad Laboratories) with image analysis performed using Image Lab (Version 6.0.0 build 25, Bio-Rad Laboratories)

Statistical analysis

All data are presented as Tukey’s box and whisker plots showing IQR, line at the median, + at the mean with whiskers ± 1.5-fold of interquartile range for at least 3 independent experiments. For all multi-group analyses, differences were determined by one-way ANOVA test followed by Holm-Sidak’s multiple comparison test. For comparisons between two groups, Mann-Whitney U test was performed. A p < 0.05 was considered to be statistically significant. All analyses were conducted using GraphPad Prism version 9.0.1 for Windows (GraphPad Software, www.graphpad.com).

MMEJ gene expression was dysregulated in high risk NB, and could be reversed by DLG2 over expression

We evaluated the expression of MMEJ genes using publicly available microarray data for four independent NB patient datasets (GSE49710, GSE16476, GSE3960, and GSE73517) obtained from the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl), and stratified according to risk status (Fig. 1a), patient survival (Fig. 1b), or 11q deletion status (Fig. 1c, d). High-risk NB showed elevated expression of PARP1 (log2 FC = 0.337, p < 0.001), FEN1 (log2 FC = 1.17, p < 0.001), and PCNA (log2 FC = 0.595, p < 0.001) with lower NBS1 expression in high risk tumors (log2 FC = -0.368, p < 0.001; Fig. 1a). The expression of these genes was also associated with poor survival, as elevated PARP1 (log2 FC = 0.454, p = 0.005), FEN1 (log2 FC = 1.09, p < 0.001), XRCC1 (log2 FC = 0.352, p = 0.04), and PCNA (log2 FC = 0.544, p < 0.001) expression were associated with higher incidence of mortality (Fig. 1b). In tumors harboring 11q deletion, FEN1 (log2 FC = 0.636, p < 0.001), XRCC1 (log2 FC = 0.549, p < 0.05), and PCNA (log2 FC = 0.378, p < 0.01) were up regulated compared to 11q-normal tumors in the GSE3960 dataset (Fig. 1c). MRE11a and NSB1 were significantly down regulated in the GSE73517 dataset (log2 FC = -1.335, p = 0.013 and log2 FC = -1.335, p = 0.013; Fig. 1d). FEN1 (log2 FC = 0.427, p < 0.05) was over expressed in the 11q deleted tumors in both 11q deleted datasets (Fig. 1c-d).

In the NB69 NB cell line (11q normal), we could show that over expression of DLG2 resulted in down regulation of PARP1 and FEN1 (log2 FC = 0.594, p = 0.001 and log2 FC = 0.818, p = 0.001) and up regulation of MRE11a (log2 FC = 0.704, p < 0.001), whereas DLG2 knockdown resulted in increased PARP1 (log2 FC = 0.637, p = 0.001), FEN1 (log2 FC = 0.925, p = 0.001) and decreased MRE11a expression (log2 FC = 0.474, p = 0.01; Fig. 1e). We verified that the changes in PARP1, FEN1, and PCNA gene expression after DLG2 over expression also decreased protein levels in the two NB cell lines, SKNBE (MYCN amplified) and SKNAS (11q deleted; Fig. 1f). To further evaluate these findings, we performed a general knockdown of dmDLG using RNAi in Drosophila melanogaster where we could confirm that loss of dmDLG resulted in increased PARP (log2 FC = 0.702, p < 0.001) and FEN1 (log2 FC = 1.170, p < 0.001) expression, as well as down regulation of MRE11a (log2 FC = 0.634, p < 0.001; Fig. 1g). Expression of MMEJ genes XRCC1 and LIG3 were not affected by DLG2 over expression or silencing in NB cells or the fly model (Fig. 1e, g).

DLG2 enhanced DNA repair in NB cells after UVC irradiation

To determine whether NB cells with or without 11q-deletion had more tendency to accumulate DNA breaks, we induced DNA breaks by exposure to UVC in combination with a forced change in DLG2 expression level. DNA integrity was assessed by exposing DLG2 transfected NB cells to UVC irradiation followed by comet assay. We could determine that silencing of DLG2 resulted in a smaller comet assay head size (68.7%, p < 0.001) and longer tail length (60.1%, p < 0.001) in 11q normal NB cells prior to UVC irradiation. Silencing DLG2 in 11q deleted cells resulted in no change in head size (15.0%, p = 0.21) but an increased tail size (33.3%, p < 0.001), indicating fewer intact DNA strands and more DNA breaks in cells with lower DLG2 levels (Fig. 2a, b). A decrease in head size (54.1%, p < 0.001) was also detected in the DLG2 over expressed cells before UVC irradiation in 11q normal NB cells. However, tail length was shown to be decreased in both 11q normal (43.2%, p < 0.001) and 11q-deleted (29.4%, p < 0.001) cells with DLG2 over expression prior to UVC irradiation, indicating a general effect on the amount of DNA breaks without inducing breaks (Fig. 2b). To determine the effect of DLG2 on DNA repair, we induced DNA breaks with UVC irradiation in 11q-deleted and 11q-normal NB cells and allowed the cells to recover for 4 hours. Using comet assay, we could determine that there was a decrease in the head size in both 11q-normal and 11q-deleted cells after irradiation in DLG2 silenced cells (20.1%, p < 0.001 and 50.4%, p < 0.001) and control samples (36.1%, p < 0.001 and 38.2%, p < 0.001); no difference was seen between the irradiated and non-irradiated samples when DLG2 was over expressed (Fig. 2a, b). There was a corresponding increase in the length of the tail in these samples; DLG2 silenced cells (83.8%, p < 0.001 and 33.9.4%, p < 0.01) and control samples (42.7%, p < 0.001 and 51.6%, p < 0.001), with no increase in the tail length of the DLG2 over expressed cells (Fig. 2b).

To further investigate the presence of double-strand DNA breaks, we investigated the presence of γ-H2AX (Ser-139, a biomarker of DNA double-strand breaks), in DLG2 silenced, control, and DLG2 over expressed NB cells lines (SKNBE (MYCN-amplified), SKNAS (11q deleted) and NB69 (MYCN and 11q normal); Fig. 2c-e respectively), 2 and 24 hours post UVC irradiation. We could determine that γ-H2AX levels were moderately repressed after 2 hours and almost completely repressed after 24 hours in cells with DLG2 over expression, indicating fewer remaining DNA breaks (Fig. 2c-e). Although, some repression of γ-H2AX was found in SKNAS and SKNBE control cells after 24 hours (Fig. 2c and d), γ-H2AX levels were constant in all cells after DLG2 silencing, thus equal amount of DNA breaks remained even 24 hours after induction (Fig. 2c-e).

To further determine the effect of UVC irradiation on DNA breakage and DLG loss we compared the DNA integrity after UVC irradiation in a Drosophila model with RNAi-silenced DLG (dmDLG knockout) to controls. We evaluated the percentage of DNA-sequences that were multiple, unique and not mapped (Fig. 2f) in control (white) and dmDLG knockout flies, 3 hours after UVC irradiation. We could ascertain that DNA from the dmDLG knockout samples had a higher percentage of non-mapped sequences compared to the control (4.33% p < 0.01; Fig. 2f). We further determined the median sequence length for the multiple, unique and not mapped sequences with dmDLG knockout flies showing a longer median read length of multiple mapped sequences (470.5bp; 45.6% increase, p < 0.05; Fig. 2g). Finally, we determined the number of multiple mapped sequence reads that contained two separate fragments from non-overlapping (greater then 10000bp gap) sequences. We could show that knockout of dmDLG resulted in an 85.4% increase in fusion sequences, (64.95% compared to the control 35.04% ± 0.5, p < 0.001; Fig. 2h).

DLG2 induced apoptosis in NB cells

We evaluated the effect of over expression of DLG2 on apoptosis in SKNBE (MYCN-amplified) and SKNAS (11q-deleted) NB cells. Overexpression of DLG2 in SKNAS cells resulted in increased mRNA expression of BAX (log2 FC = 1.47, p < 0.001) as well as decreased BCL2 expression (log2 FC = 0.713, p < 0.001); DLG2 silencing resulted in decreased mRNA expression of BCL2 (log2 FC = 0.744 p = 0.001) with no alteration in BAX expression (Fig. 3a, b). The ratio of BAX/BCL2 gene expression was determined and shown to be high in cells with DLG2 over expression (FC = 4.16, p < 0.01), with no alteration for DLG2 silenced cells (FC = 0.329, p = 0.931; Fig. 3c). Caspase 3/7 activation was measured over time to determine apoptosis activity. There was no difference in caspase 3/7 activation between control cells and DLG2 transfected cells 24 hours post transfection; there was an increase in activation at 30 hours (27.8%, p < 0.001) and 54 hours (15.6%, p < 0.001), with no difference in the level of activation again at 70 hours (Fig. 3d). We could confirm that BAX gene expression changes after DLG2 over expression resulted in increased levels of BAX protein in both SKNBE and SKNAS cells, and that BCL2 protein levels were lowered in SKNAS cells (Fig. 3e). DLG2 over expression resulted in an increase in p53 and Ser46 phosphorylated p53 levels in both SKNBE (83.1%, p < 0.05 and 23.9%, p < 0.05; Fig. 3f-g) and SKNAS (45.9%, p < 0.01 and 53.4%, p < 0.01; Fig. 3h-i) cell lines. These results showed that DLG2 over expression induce apoptosis in NB cells.

High DLG2 levels enhanced CHK1 phosphorylation after UVC irradiation in NB cells

To determine the effect of DLG2 on DNA repair in NB, we investigated DNA damage response pathways (DDR) in NB cell lines after DLG2 silencing or DLG2 over expression and UVC irradiation. We could show a strong activation of CHK1-S317 in all cells 2 hours after UVC irradiation, with the weakest activation in DLG2 silenced cells. This activation was lost in all cells after 24 hours recovery, except in those with DLG2 over expression (Fig. 4a, b). There was no difference in basal CHK1 expression. Subsequent evaluation of CHK2-T68 phosphorylation demonstrated expression in DLG2 silenced and control cells after 2 hours of recovery for SKNBE and SKNAS but not detectable in NB69, which was lost after 24 hours of recovery; basal expression of CHK2 was unaffected in SKNBE cells, but decreased in the SKNAS and NB69 cell lines with DLG2 over expression (Fig. 4a, c). No difference in the total amount of ATM or DNA-PKCs could be determined, which were neither affected by DLG2 levels nor the time of recovery after UVC irradiation (Fig. 4a). We quantified the phosphorylation of S317 CHK1 and T68 CHK2 and could show that phosphorylation of CHK1-S317 decreased over time in the DLG2 silenced and the control cells (9.5%, p < 0.01 and 7.5%, p < 0.05; Fig. 4b). DLG2 over expression resulted in an increase in S317 CHK1 (9.8%, p < 0.01; Fig. 4c). The phosphorylation of T68 CHK2 decreased over time in the DLG2 silenced and the control cells (27.5%, p < 0.05 and 28.7%, p < 0.05; Fig. 4c) with no difference over time in DLG2 over expressed cells.

Previous attempts have been made to determine the illusive tumor suppressor gene located on 11q, with varying success [6]. It has previously been reported that there are two tumor suppressor genes located on chromosome 11q that could act in a combined haploinsufficient manner [27]. Here, we investigated the convergence of DLG2 (11q14.1) and the 11q23 deleted candidate TSGs within the DNA damage repair pathway. Previous studies have shown that some of the 11q23 SRO candidate TSGs function within this pathway [35–37], the roll of DLG2 in DNA repair has only been previously suggested with bioinformatics [27]. We have shown changes in the expression of DNA repair genes in primary NB samples with unfavorable prognosis and 11q deletion (Fig. 1a-d). As patients with 11q deleted NBs are classified as high risk, with poor survival and a high number of DNA breaks and chromosomal alterations, we investigated how DLG2 alters these genes of interest.

We have recently shown that there are two isoforms of DLG2 expressed in NB, isoform 2 (ISO2) and isoform 7 (ISO7); and that DLG2 isoform 7 is lost in advanced staged NB [38]. Here, we shown that DLG2-ISO7 interrupts the expression of genes associated with the DNA damage repair machinery, i.e. PARP1, FEN1, PCNA as well as induce elevated MRE11a expression (Fig. 1e), another gene located in chromosome region 11q. Inhibition of PARP1 [39, 40] and PCNA [41] have previously been investigated in NB, while FEN1 is known to be up regulated in tumors with MYCN amplification [42]. To further investigate these findings, we silenced the fly orthologue gene dmDLG in a Drosophila model, which subsequently resulted in increased dmFEN and dmPARP, thereby reproducing the altered PARP1 and FEN1 expression found in NB patient samples and NB cell lines (Fig. 1g). Subsequently we silenced dmDLG in the fly and irradiated with UVC which resulted in increased DNA damage in the form of an increase in the number of sequences that could not be mapped, indicative of low genome integrity (Fig. 2f). Furthermore, the increased median length of multiple mapped sequence in the DNA from dmDLG silenced flies compared to control indicated that there were likely fusion-sequences present (Fig. 2g). This was confirmed by looking at the number of multiple mapped sequences that were comprised of 2 distinct fragments (Fig. 2h). The loss of dmDLG combined with UVC irradiation activated low fidelity DNA repair, taken with the previous results, we show that loss of dmDLG results in upregulation of MMEJ.

To further investigate the effects that DLG2 has on DNA repair, we induced DNA breaks by exposing transfected cells to UVC irradiation and allowing the cells to recover. Here we could show that DLG2 maintained genome stability and integrity, whereas loss of DLG2 resulted in high fragmentation without the extra stimulus of the UVC irradiation (Fig. 2a-b). This increase in DNA fragmentation may be the direct result of aberrantly activated endonuclease activity such as that found in FEN1 [43] or loss of the G2/M checkpoint. After UVC stimulus, the NB cells with DLG2 loss or silencing displayed a highly unstable genome with persisting DNA strand breaks. To show that we induced double-strand breaks, we investigated phosphorylation of S139 H2AX. Here we could show that double-strand breaks were still present 24 hours after UVC irradiation when DLG2 was down regulated (Fig. 2c-e), indicating that DNA repair had not yet occurred or that FEN1-mediated genome fragmentation was actively occurring. It has previously been shown that loss of DLG2 removes the G2/M DNA damage checkpoint [27], maintenance of which requires CHK1 activation [44]. Consistent with this, we show that restoration of DLG2 expression maintained S317 CHK1 phosphorylation, whereas there was a decrease in phosphorylation in the DLG2 silenced and control cells (Fig. 4). The activation of S317 CHK1 occurs primarily through ATR but it can also occur through ATM [45]. Interestingly, there was phosphorylation of T68 CHK2 at 2 hours in the DLG2 silenced and control cells but not in the DLG2 over expressed cells. The protein was subsequently dephosphorylated after 24 hours recovery in all experiments (Fig. 4). The phosphorylation of T68 CHK2 occurs primarily by ATM, indicating that DLG2 and ATM operate within different pathways [45]. To further complicate the mechanism, DLG2 over expression resulted in down regulation of total CHK2 expression in all cell lines except SKNBE, a MYCN amplified cell line. These results and the somewhat redundant nature of CHK1 and CHK2 imply that DLG2 pushes the cells towards CHK1 and away from CHK2 regulation.

We subsequently investigated the effects of DLG2 on apoptosis. We could show that elevated expression of DLG2 resulted in increased total p53, the effect of which was seen in the expression of the transcriptional targets BAX and BCL2 (Fig. 3). The increased ratio of BAX/BCL2 increases the permeability of the mitochondrial membrane as BCL2 cannot inhibit BAX pore formation and leads to apoptosis [46]. We could also detect an increase in pS46 phosphorylated p53 level, which is generally associated with severe genotoxic stress and often leads to apoptosis [47]. Genotoxic stress is present in MYCN amplified NB tumors [39] as well as 11q deleted tumors[7]. We confirmed that there was a transient increase of the executioner caspases 3/7 post DLG2 transfection, showing that the pS46 p53 phosphorylation result in apoptosis in NB. ATM is one of the essential kinases that phosphorylates pS46 p53 [23, 36], in this case DLG2 might cooperate with ATM by increasing BAX expression and allow for pS46 p53 mediated binding, a key step for transcription independent apoptosis [48]. Further studies into the coalescence of candidate 11q tumor suppressor genes are required to fully understand the causes of increased relapse and poor survival in 11q deleted NB patients.

We have shown that DLG2 over expression increases the ability to sense genotoxicity, reduce DNA replication speed, as well as increase pS46 p53 phosphorylation to induce apoptosis in both 11q deleted and MYCN amplified NB cells. This adds evidence that DLG2 is a NB tumor suppressor gene. Furthermore, DLG2 maintains NB cells at the G2/M checkpoint by S317 CHK1 phosphorylation, independent of ATM.

MMEJ: Microhomology mediated end joining; NB: Neuroblastoma; NHEJ: Non-homologous end joining; HR: Homologous Repair; SRO: Smallest region of overlap; S46: Serine 46; T68: Threonine 68; CHK; Checkpoint kinase;

Ethics approval and consent to participate

Only primary tumor data from publically available sources were used in this study.

Consent for publication

All authors give consent for the publication of the manuscript.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by grants from the Swedish Childhood Cancer Fund, Jane and Dan Olsson Foundation and Assar Gabrielsson Fund. The funders were not involved in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Open access funding provided by University of Skövde.

Authors' contributions

KE generated conception and designed this study and provided technical and material support. SK developed the methodology, performed the assays, analyzed and interpreted the data. HdW performed the bioinformatic analysis. SK and KE organized the data and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank the Swedish Childhood Cancer Fund, Jane and Dan Olsson foundation, Assar Gabrielssons Foundation and University of Skövde for financial support.

Authors' information (optional)

Affiliations

Translational Medicine, School of Health Sciences, University of Skövde, Skövde, Sweden

Simon Keane & Katarina Ejeskär

School of Bioscience, Systems biology research center, University of Skövde, Skövde, Sweden

Hendrik A. de Weerd

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DLG2 impairs dsDNA break repair and maintains genome integrity in neuroblastoma

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