RNA 5-methylcytosine status is associated with DNMT2/TRDMT1 nuclear localization in osteosarcoma cell lines

Highlights • Selected phenotypic features of three osteosarcoma (OS) cell lines were evaluated.• Redox disequilibrium promoted sustained AKT and ERK1/2 activation.• Redox imbalance modulated cell death pathways in OS cells.• Nuclear levels of TRDMT1 methyltransferase were associated with RNA methylation.• A novel marker for predicting therapy response in OS patients is proposed.


Introduction
Osteosarcoma (OS) is the most common primary bone tumor affecting mostly children and adolescents [1]. The annual rate of global OS incidence is 3.4 per million people and the 5-year survival rate is 60-70% [2,3]. OS is highly metastatic and has a high risk of recurrence after treatment [4]. Development of OS has been associated with the mutations of tumor suppressors, such as TP53, RB1, REQL4, and INK4a and/or deregulation of PI3K/mTOR, RANKL/NF-κB, TGFβ, and IGF pathways [5]. However, agents targeting these pathways have not achieved successful clinical outcomes [5]. The heterogeneous profile, the unbalanced karyotype, and changes in immune dynamics make conventional treatment regimens of OS less efficient [6][7][8]. Therefore, it is important to understand the mechanisms underlying the development of OS to find new alternative molecular approaches for early diagnosis and therapy.
For in vitro studies of OS, U-2 OS, SaOS-2, and MG-63 cell lines are frequently used as experimental models [9][10][11]. These are one of the most popularly available OS cancer cell lines that share many similarities [12], hence providing a platform for parallel comparisons. Despite the well-known similarities, there are, however, differences between these cell lines. For example, U-2 OS, SaOS-2, and MG-63 cells possess a different degree of differentiation [13]. SaOS-2 cells show rather a mature phenotype, while U-2 OS cell line is negative for almost all osteoblastic markers. Interestingly, MG-63 cells have the most heterogeneous profile, as they possess both mature and immature osteoblastic features [13]. Additionally, U-2 OS cells are positive for cartilage marker, collagen IV type, which is only expressed in very early differentiation stages but not by mature osteoblasts [13]. Therefore, U-2 OS cells are frequently classified as fibroblastic [14,15]. Moreover, the large panel of OS cell lines was genetically described [16] and proteome comparison of U-2 OS and SaOS-2 cells has been performed [17]. The comparative studies revealed 89 up-regulated and 86 down-regulated proteins in SaOS-2 cells compared to U-2 OS cells [17].
Furthermore, these cell lines are used as a reference model in studies that aim to develop new drugs, find druggable targets and gain insight into drug metabolism [11,[18][19][20][21]. Recently, anticancer epigenetic drugs targeting RNA methylation are becoming a hot topic in cancer research, including OS [22][23][24]. Interestingly, it has been shown that knockout (KO) of DNMT2/TRDMT1 gene (encoding tRNA-aspartic acid methyltransferase 1) compromised homologous recombination (HR) process and prevented drug resistance in OS cells [25,26]. Moreover, our previous studies have shown that U-2 OS cells lacking DNMT2/TRDMT1 gene exhibit changes in DDR mechanism, telomere length, retrotransposon activity, and IFN-β-mediated response leading to cellular and genetic heterogeneity that modulate cancer cell responses to chemotherapy [27]. These new findings indicate a pivotal role of TRDMT1 in OS therapy, motivating us to investigate TRDMT1-mediated RNA m 5 C modification in other OS cell lines. Moreover, the comparative studies of DNMT2/TRDMT1-based response and the differences in the activity of signaling pathways between U-2 OS, SaOS-2 and MG-63 cells have not yet been revealed. Identifying the differences between these cell lines may help to understand the diverse and even opposite outcomes in response to the same pharmacological treatment. Moreover, specific determination of the traits of U-2 OS, SaOS-2, and MG-63 cells can give a hint, which of the cell line is the most relevant to predicting efficient treatment in the clinic.
In this study, we evaluated selected phenotypic features of three osteosarcoma cell lines, namely U-2 OS, SaOS-2 and MG-63 cells and focused on cell death pathways, redox status, the activity of proliferation-associated pathways, DNA damage response, DNMT2/ TRDMT1 methyltransferase-based effects and RNA 5-methylcytosine status. We documented that redox disequilibrium may promote sustained AKT and ERK1/2 activation thus affecting cell death pathways in OS cells. Furthermore, nuclear TRDMT1 levels may be associated with the status of RNA 5-methylcytosine that may be considered as a novel molecular marker for predicting therapy response in OS patients.

Apoptosis
Phosphatidylserine externalization (an apoptotic biomarker) was evaluated using Muse® Cell Analyzer and Muse® Annexin V and Dead Cell Assay Kit following the manufacturer's instructions (Luminex Corporation, Austin, TX, USA). Representative dot plots are shown.

Oxidative stress
Changes in intracellular superoxide levels were analyzed using Muse® Cell Analyzer and Muse® Oxidative Stress Kit (Luminex Corporation, Austin, TX, USA) as previously described [23]. Briefly, superoxide-positive and superoxide-negative subpopulations were determined. Representative histograms (%) are presented.

Nitric oxide levels
Nitric oxide levels were evaluated using Muse® Cell Analyzer and Muse® Nitric Oxide Kit (Luminex Corporation, Austin, TX, USA) as described elsewhere [28]. Briefly, nitric oxide specific fluorogenic probe DAX-J2 TM orange and a death marker, 7-aminoactinomycin D (7-AAD) were used to discriminate between the production of nitric oxide in live (7-AAD-negative) and dead (7-AAD-positive) cells. Representative dot plots are presented.

PCR amplification and Sanger sequencing of DNMT2/TRDMT1 mRNA isoforms
2000 ng of total RNA were reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). The PCR assay encompassed amplification of eight fragments covering the DNMT2/TRDMT1 mRNA reference sequence of four isoforms (Table S1). Two pairs of PCR primers for each of four isoforms (Fig. S1) were designed using the Primer3 software [30,31] and synthesized by Thermo Fisher Scientific (Waltham, MA, USA). Amplification was performed using hot start Taq DNA polymerase (Qiagen, Hilden, Germany). After an initial step of 95 • C for 15 min, a touchdown PCR procedure (TDPCR) was applied. The initial TDPCR stage included 12 steps of annealing temperature decreases of 1.0 • C in the range from 67 • C down to 56 • C. The second stage of TDPCR included 30 PCR cycles with annealing at 55 • C. Each cycle of amplification included 30 s of denaturation at 94 • C, 1 min of annealing, and 30 s of extension at 72 • C. Amplification was ended with the final extension step at 72 • C for 7 min. PCR amplifications were performed on the VeritiPro Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). Amplified cDNA samples were run in two percent agarose gel stained with ethidium bromide for the evaluation of the quality of amplified cDNAs. PCR products were sequenced using PCR primers and the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA). The manufacturerrecommended cycle sequencing program was applied with the primer annealing of 59 • C and 4 min of extension at 60 • C. Sequencing of PCR products was performed on the VeritiPro Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing products were cleaned with the BigDye XTerminator™ Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) and electrophoresed on the ABI3500xl genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA).
Obtained sequencing traces were evaluated for quality and subjected to variant genotyping using FinchTV 1.4.0 (Geospiza Inc. Seattle, WA, USA). Further investigations included trace sequence alignment against reference sequences using GENEDOC software [32] to check for the variation among investigated cDNA samples. Obtained sequence traces were also aligned against a reference sequence of the human genome using the blastx option of the NCBI website to check for their homology to the translated sequences encoding DNMT2/TRDMT1 protein isoforms. GENEDOC software was used for cDNA sequence translation of reported variants of DNMT2/TRDMT1 mRNA.

In silico analysis of potential DNMT2/TRDMT1 post-translational modifications
To test possible post-translational modifications of DNMT2/ TRDMT1 freely available computational tool Scansite 4.0 (http://scansite.mit.edu) was used. The Scansite program uses scores calculated from position-specific score matrices (PSSM) to search for motifs. The settings of high stringency were selected. The high stringency setting is the most restrictive and reports the motif identified in the query sequence that falls within the top 0.2% of all matching sequences contained in the vertebrate subset of SwissProt proteins. DNMT2/TRDMT1 amino acid sequence was obtained from Uniprot (https://www.uniprot.org/), accession number O14717.

Statistical analysis
The results represent the mean ± SD of at least three independent experiments. Box and whisker plots with median, lowest, and highest values were also used. Statistical analysis was performed using the GraphPad Prism 8 software. Comparison between U-2 OS, SaOS-2 and MG-63 cell lines were performed with one-way ANOVA followed by Dunnett's test. Differences between SaOS-2 and MG-63 cells were revealed using one-way ANOVA and Tukey's multiple comparison test. The differences in RNA methylation between all three cell lines were assessed using Student's t-test. P < 0.05 was considered statistically significant.

Comparison of the activity of proliferation-related signaling pathways in U-2 OS, SaOS-2, and MG-63 cells
The in vitro cancer model is a major tool to understand the molecular basis of cancer biology for appropriate selection of potential anticancer agents and the investigation of their effectiveness. Thus, it is essential to provide comprehensive characterization of widely used cancer cell lines. In the present study, three osteosarcoma (OS) cell lines, namely U-2 OS, SaOS-2, and MG-63 cells, were compared in terms of cell death pathways, redox homeostasis, the activity of selected proliferationassociated signaling pathways, DNA damage response, telomere maintenance and DNMT2/TRDMT1-based responses that might be potentially important for improving existing and developing new anticancer strategies.
Chemotherapy-induced apoptosis is widely used anticancer approach, however, cancer cells can differently respond to apoptotic stimuli and acquire resistance to chemotherapeutic agents [33]. Therefore, we first compared the susceptibility to apoptotic and necrotic cell death of three OS cell lines in standard growth conditions (Fig. 1A). Slight but statistically significant differences in apoptotic and necrotic parameters were noticed (Fig. 1A, p < 0.01, p < 0.001). MG-63 cells were the least prone to apoptotic cell death compared to U-2 OS and SaOS-2 cells (Fig. 1A, p < 0.01). In contrast, SaOS-2 cells were the most susceptible to necrotic cell death compared to other OS cells (Fig. 1A, p < 0.001).
Cancer cells are characterized by elevated production of reactive oxygen species (ROS) mainly due to increased metabolic rates [34]. The role of ROS in cancer biology is rather complex [35,36]. A moderate increase in ROS levels has been shown to promote pro-tumorigenic signaling, enhance tumor development and progression, drive DNA damage and genome instability, and modulate drug resistance [34,37]. However, ROS are also capable of triggering programmed cell death by activating signaling pathways and modulation of oxidative stress is considered as an anticancer strategy [34,[37][38][39]. As different , namely superoxide-negative subpopulation (blue) and superoxide-positive subpopulation (red). Representative histograms are shown. Bars indicate SD, n = 3, *** p < 0.001 compared to U-2 OS cells (ANOVA and Dunnett's a posteriori test). Red color of asterix corresponds to superoxide-positive cell subpopulation. (C) Nitric oxide levels were analyzed using an DAX-J2 TM orange probe and flow cytometry. Representative dot plots are shown. Bars indicate SD, n = 3, ** p < 0.01, *** p < 0.001 compared to U-2 OS cells (ANOVA and Dunnett's a posteriori test). Black color of asterix corresponds to dead cell subpopulation that produces nitric oxide. (D) ERK1/2 and AKT activity (phosphorylation status) was analyzed using Muse® Cell Analyzer. Representative dot plots are shown. Bars indicate SD, n = 3, *p < 0.05, ** p < 0.01 compared to U-2 OS cells (ANOVA and Dunnett's a posteriori test). Red color of asterix corresponds to dual activation of ERK1/2 and AKT pathways. susceptibility to cell death was documented in three OS cell lines (Fig. 1A), we were interested then to analyze if this observation can be related to diverse rates of superoxide production (Fig. 1B). Indeed, a slight but statistically significant difference in ROS production in SaOS-2 cells compared to U-2 OS and MG-63 cells (Fig. 1B, p < 0.001) can reflect the susceptibility of SaOS-2 cells to necrotic cell death (Fig. 1A).
Next, we have focused on the analysis of nitric oxide (NO • ) levels as elevated pools of nitric oxide are commonly observed in cancer cells [40]. However, NO • may also have a role in sensitizing the cancer cells to chemotherapeutic treatment [41]. SaOS-2 cells were characterized by the highest levels of nitric oxide compared to other OS cells (Fig. 1C, p < 0.001) that can be also correlated with the highest levels of superoxide (Fig. 1B) and the susceptibility to necrotic cell death (Fig. 1A).
ROS may modulate proliferation-related signaling pathways such as PI3K/AKT and extracellular signal-regulated kinase (ERK) pathways [42][43][44]. Dysregulation of PI3K/AKT pathway has been associated with pathological processes of OS, including proliferation and invasion, cell cycle progression, apoptosis, angiogenesis, metastasis, and chemoresistance [45]. Similar as PI3K/AKT, ERK1/2 pathway was related to proliferation [46], apoptosis [47] and metastasis [48]. Therefore, we analyzed then the levels of phosphorylated forms of AKT and ERK1/2 in three OS cell lines (Fig. 1D). SaOS-2 cells were characterized by slightly higher levels of both phosho-AKT and phospo-ERK1/2 compared to other OS cells (Fig. 1D, p < 0.01). Our data are in agreement with the study showing that the PI3K-AKT-mTOR pathway could also be a prodeath signal, especially in the context of necrotic cell death [49,50]. Moreover, it has been shown that ERK activation potentiated cisplatininduced cell death in SaOS-2 cells [51].
Overall, this may suggest that ROS and NO • production in SaOS-2 cells may promote sustained AKT and ERK1/2 activation thus affecting cell death pathways. Interestingly, SaOS-2 cells have previously been suggested to be more resistant to chemotherapy than other OS cell lines [52]. Therefore, the use of oxidative stress-induced drugs and NO • donors could be a possible strategy to sensitize SaOS-2 cells to chemotherapy-associated cell death.

Differences in the DNA damage response (DDR) in OS cell lines
Genomic instability, a consequence of defective DNA repair pathways or misregulated cell-cycle checkpoints, is one of enabling characteristics of cancer cells [53,54]. The components of DDR can be considered as druggable targets in anticancer strategies [55]. Therefore, we checked the differences in the DDR between OS cell lines. Our analysis indicated very slight differences in ATM activation and dual ATM and γH2AX activation in SaOS-2 compared to other OS cells ( Fig. 2A).
We evaluated then the differences in the expression of selected factors involved in DNA repair mechanism, namely the levels of RAD51, RAD52, and XRCC1 proteins. Interestingly, RAD51 gene knockdown (KD) has been shown to sensitize OS cells to chemo-and radiotherapy [56], while RAD52 is involved in mitotic repair synthesis at telomeres [57]. Chen et al. have reported that U-2 OS-TRE cells lacking TRDMT1 gene impairs the localization of RAD51 and RAD52 to the sites of ROSinduced DNA damage [25]. Moreover, our previous studies have shown that treatment with etoposide caused changes in the RAD51 and RAD52 expression levels in U-2 OS cells with DNMT2/TRDMT1 gene KO sensitizing them to the drug treatment [27]. Based on our analysis, the protein levels of RAD51 and XRCC1 were comparable in three OS cell lines (Fig. 2B). Interestingly, RAD52 is expressed in the U-2 OS cell line, whereas no expression of RAD52 was noticed in MG-63 and SaOS-2 cell lines (Fig. 2B). Our analysis is in contrast with the previous studies showing the presence of RAD52 in SaOS-2 and MG-63 cells at the low levels [58].
Telomeres prevent chromosome ends from inducing DNA damage and end-to-end fusion that results in rearrangements [59]. Although the length of the telomere is commonly maintained by telomerase, cancer cells also employ a recombination-based mechanism known as alternative lengthening of the telomeres (ALT) [59]. Of the OS cell lines studied, U-2 OS and SaOS-2 have been characterized as ALT-positive [60,61], while MG-63 as telomerase-positive and ALT-negative [62,63]. Since many DNA repair proteins, including RAD52, have been implicated in the ALT mechanism [57,58], the lack of RAD52 expression in SaOS-2 cells seems to be surprising (Fig. 2B). As the OS cell lines use distinct mechanisms to maintain the telomeres, we evaluated the differences in their telomere length. Based on flow cytometry, we could indicate that the involvement of different mechanisms leads to differences in telomere length in OS cell lines (Fig. 2C). Our data are in agreement with the Southern blot-based studies showing following telomere length ranking U-2 OS cells (13.7 kbp) > SaOS-2 cells (7.6 kbp) > MG-63 cells (5.8 kbp) [64].
A growing body of evidence suggest that HR process cooperates with components of the shelterin complex [65]. The activity of RAD52 and other HR factors, together with shelterin components TRF2 and TIN2 are required for telomeric D-loop formation [65]. TRF1 and TRF2 are two out of six shelterin complex components and participate in both telomerase-dependent telomere maintenance and ALT mechanism [66,67]. Additionally, binding by TRF1 and TRF2 is essential to protect telomeres against DDR and DNA repair factors [68]. Therefore, we compared then the expression of TRF1 and TRF2 between OS cells. Based on our analysis, we observed a slightly higher expression of TRF1 and TRF2 in U-2 OS cells compared to SaOS-2 and MG-63 cells (Fig. 2D).

The nuclear levels of DNMT2/TRDMT1 methyltransferase are associated with the RNA 5-methylcytosine modification status
RNA methylation in cancer progression has become increasingly prominent. RNA methylation occurs most often at the C5 position of cytosine (RNA:m 5 C) [69]. RNA:m 5 C is regulated by m 5 C-methyltransferases, namely tRNA-specific methyltransferase (DNMT2/ TRDMT1) family members and NOL1/NOP2/SUN domain (NSUN) family members. NSUN methyltransferases include several members, namely NSUN1-NSUN7 [70,71]. Out of all NSUN members, NSUN2 possesses the most prominent role in cancer progression [72]. Similarly to NSUN2, DNMT2/TRDMT1 is a multifunctional enzyme that participates in the regulation of cancer cell proliferation, migration, and DDR [23,25,27,73]. Furthermore, the dysregulation of the modification is closely related to human tumor malignancies [74]. Although, RNA:m 5 C plays a crucial role in the diagnosis and prognosis of numerous cancers [75][76][77][78], our understanding of the regulation and function of RNA 5methylcytosine (RNA:m 5 C) modification in OS remains limited. Therefore, we first evaluated the protein levels of DNMT2/TRDMT1 in three OS cell lines (Fig. 3A). Western blot analysis indicated the highest levels of DNMT2/TRDMT1 protein in U-2 OS cells and the lowest levels in SaOS-2 cells, respectively (Fig. 3A).
Since DNMT2/TRDMT1 can be located both in cytoplasmic and nuclear compartments [79][80][81], we have then investigated the differences in the cytoplasmic and nuclear pools of DNMT2/TRDMT1 between U-2 OS, SaOS-2 and MG-63 cells. Based on our analysis, MG-63 cells were characterized by the highest levels of nuclear DNMT2/ TRDMT1 compared to other OS cells (Fig. 3B, p < 0.001). U-2 OS cells with the highest total levels of DNMT2/TRDMT1 (Fig. 3A) were also characterized by the highest levels of cytoplasmic fraction of DNMT2/ TRDMT1 compared to other OS cells (Fig. 3B). SaOS-2 cells, with the lowest levels of total DNMT2/TRDMT1 (Fig. 3A), possessed also the lowest levels of cytoplasmic and nuclear DNMT2/TRDMT1 (Fig. 3B, p < 0.001). Additionally, we evaluated the nuclear and cytoplasmic fractions of NSUN2 (Fig. 3B). SaOS-2 cells were characterized by the lowest levels of cytoplasmic and nuclear NSUN2 compared to other OS cells (Fig. 3B).
Next, we have evaluated the differences in the levels of 5-methylcytosine in RNA of OS cell lines (Fig. 3C). The highest methylation status was observed in MG-63 cells, whereas the lowest levels of 5-methylcytosine were noticed in SaOS-2 cells (Fig. 3C, p < 0.001).
Interestingly, it has been previously shown that DNMT2/TRDMT1 may confer resistance to anticancer therapy [25,26]. Therefore, the levels of DNMT2/TRDMT1 can be considered as a novel predictive marker of anticancer drug response, especially in the context of genotoxic drugs.

Changes in the DNMT2/TRDMT1 localization may be mediated by post-translational modifications
As the localization of DNMT2/TRDMT1 significantly differs between the cell lines studied, we have next tried to determine the reason for this diversity. It has been previously shown that DNMT2/TRDMT1, which is typically present in nucleus, relocalized to the cytoplasm under stress conditions [82]. Therefore, we check whether this is due to the impaired nuclear architecture that may affect nucleus function. The association between higher lamin B1 expression levels and nuclear dysmorphisms in OS cell lines has been already suggested [83]. Moreover, it has been shown that lamin A interacts with the m 6 A methyltransferases, METTL3 and METTL14 in nuclear speckles [84]. Therefore, we evaluated the differences in the levels of lamin A/C and lamin B1. However, our analysis indicated that SaOS-2 and MG-63 cells (with the lowest and highest levels of nuclear DNMT2/TRDMT1, respectively) possess similar levels of lamin A/C and lamin B1, which is also higher than in U-2 OS cells (Fig. 4A).
Since alternative splicing results in multiple transcript variants encoding different DNMT2/TRDMT1 isoforms (based on https://www. uniprot.org/uniprot/O14717), we tested whether the different DNMT2/TRDMT1 isoforms may show distinct localization patterns. Our analysis, based on sequence alignment including variant A (accession number NM_004412) of DNMT2/TRDMT1 mRNA and cDNA samples amplified using primers specific to four isoforms (D, E, F and G), showed no differences (Fig. S2). Furthermore, we performed a protein sequence alignment showing the homology of the tested samples at amino acid levels without differences as well (Fig. S3). Therefore, we decided to predict whether the cytoplasmic and nuclear localization of DNMT2/TRDMT1 might be caused by posttranslational modifications of DNMT2/TRDMT1. To test this, we used different bioinformatics tools, however, our interest focused on results obtained using the http://scansite.mit.edu. Scancite identifies short protein sequence motifs that are likely to be phosphorylated or mediate specific interactions with protein or phospholipid ligands [85]. Interestingly, high-resolution computational analysis revealed TRDMT1 as a potential phosphorylation target for ERK1 (MAPK3) at P361, PLCg (PLCG1) at Y266, and DNA-PK (PRKDC) at S327 (Fig. 4B and C). All predicted phosphorylation target sites are present in all isoforms studied. Furthermore, scanning analysis predicted DNMT2/TRDMT1 colocalization with PLCg in the cytoplasm, while with ERK1 and DNA-PK in the nucleus (Fig. 4C). However, this analysis needs further validation using inhibitors and/or silencing of potential candidates.
In conclusion, we have shown for the first time that U-2 OS, SaOS-2, and MG-63 OS cell lines differ in the activity of proliferation-related signaling pathways, redox equilibrium, cell death pathways and DNA damage response. Moreover, we observed that MG-63 cells, which possess the highest levels of nuclear DNMT2/TRDMT1, exhibit the most abundant methylated RNA at the m 5 C position. Additionally, we hypothesize based on in silico analysis that the distinct cellular localization of DNMT2/TRDMT1 may be a result of post-translational modifications. Altogether, detailed characterization of three OS cell lines provided in the present study may be helpful for a better understanding of OS biology and identifying new targets for OS treatment.

Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Maciej Wnuk reports financial support was provided by National Science Centre Poland.