The RNA‐binding protein La/SSB associates with radiation‐induced DNA double‐strand breaks in lung cancer cell lines

Abstract Background Platinum‐based chemotherapy and radiotherapy are standard treatments for non‐small cell lung cancer, which is the commonest, most lethal cancer worldwide. As a marker of treatment‐induced cancer cell death, we have developed a radiodiagnostic imaging antibody, which binds to La/SSB. La/SSB is an essential, ubiquitous ribonuclear protein, which is over expressed in cancer and plays a role in resistance to cancer therapies. Aim In this study, we examined radiation‐induced DNA double strand breaks (DSB) in lung cancer cell lines and examined whether La/SSB associated with these DSB. Method Three lung cancer lines (A549, H460 and LL2) were irradiated with different X‐ray doses or X‐radiated with a 5 Gy dose and examined at different time‐points post‐irradiation for DNA DSB in the form of γ‐H2AX and Rad51 foci. Using fluorescence microscopy, we examined whether La/SSB and γ‐H2AX co‐localise and performed proximity ligation assay (PLA) and co‐immunoprecipitation to confirm the interaction of these proteins. Results We found that the radio‐resistant A549 cell line compared to the radio‐sensitive H460 cell line showed faster resolution of radiation‐induced γ‐H2AX foci over time. Conversely, we found more co‐localised γ‐H2AX and La/SSB foci by PLA in irradiated A549 cells. Conclusion The co‐localisation of La/SSB with radiation‐induced DNA breaks suggests a role of La/SSB in DNA repair, however further experimentation is required to validate this.

DNA-damaging treatment approaches may have curative potential, it is mainly treatment resistance that limits their effectiveness.
Although an intent of cytotoxic anti-cancer treatment is cancer cell death, cancer cells surviving the assault may adopt altered cellular states, which have reduced proliferative potential, but which may also exert persisting deleterious effects within the immediate microenvironment and more extensively via elaboration of exosomes for example. 6 Although manifold and complex, among the pro-survival mechanisms contributing to treatment resistance after DNA damage are the induction of anti-apoptotic signalling pathways 7 and accelerated DNA repair. 8 But there are also instances of lower fidelity DNA repair, which may promote genome instability and adaptive mutations. 6 We have been interested to understand the contribution that cancer cell death makes to effective radiotherapy, chemotherapy and immunotherapy. To that end, as an in vivo marker of cancer cell death, we have developed a novel radiodiagnostic monoclonal antibody (mAb) for imaging, which is called chimeric DAB4 (chDAB4) and which is trademarked as APOMAB ® . 9,10 The chDAB4 mAb has entered a phase 1 clinical imaging trial in advanced NSCLC patients who will receive first-line chemo-immunotherapy and/or radiotherapy (Australian and New Zealand Clinical Trials Registry No. 12620000622909). The chDAB4 mAb is specific for the essential, exceedingly abundant and ubiquitously expressed 46 kDa RNA-binding protein, La/SSB. 11 The lupus-associated (La) antigen has the HUGO Gene name of Sjögren Syndrome B (SSB) and is also known as La-related protein 3 (LARP3). Based on an earlier preclinical imaging study, 9 the clinical rationale for this radioimmunodiagnostic approach is that patients who respond to the lung cancer treatment will demonstrate significant tumour uptake of radiolabelled chDAB4 whereas it is presumed that non-responding patients will have treatment-resistant disease.
La/SSB is over expressed in malignancy 12 and in clinical samples including of lung cancer, 13 cervical cancer, 14 head and neck squamous cell carcinoma (HNSCC), 15,16 chronic myeloid leukaemia (CML), 17 polycythaemia rubra vera and primary myelofibrosis. 18 The chDAB4 mAb only binds the La/SSB protein in dead cancer cells. During apoptotic tumour cell death in vitro, the La/SSB protein translocates from nucleus to cytoplasm, and as necrosis develops with loss of cell membrane integrity, the La/SSB protein becomes available for antigenspecific antibody binding in the dead tumour cells. 12,13,[19][20][21][22] Moreover, after DNA-damaging anti-cancer treatments such as some cytotoxic chemotherapy drugs or ionising radiation, the binding of specific antibodies to La/SSB in dead tumour cells is even greater because of two major effects. First, treatment-induced tumour cell death creates more La/SSB binding targets. Second, for poorly understood reasons, the per cell binding of La/SSB-specific antibodies to dead tumour cells also increases. 12,19,20,22 In an earlier study, after cytotoxic drug treatment of tumour cells, chromatin-associated La/SSB was shown to increase and to co-localise with double strand breaks (DSB) using immunofluorescence. 12 In vivo, apoptotic cells, which are created at the rate of a million cells a second, are never evident because they are cleared highly efficiently before there is time for them to become necrotic. In vivo, after chemotherapy is given to tumour-bearing mice, necrotic tumour cells are cleared inefficiently (unlike dead normal cells) and thus are available for in vivo binding by La/SSB-specific antibody. 21 Although specific aspects of the oncogenic role of La/SSB overexpression are being uncovered, 14,15,[23][24][25] it is likely that La/SSB plays a multi-functional role in malignancy 26 as it has been shown to perform physiologically. La/SSB is estimated to exist as 20 Â 10 7 copies per cell, which makes it as abundant as a ribosomal protein. 11 The RNAbinding functions of La/SSB are critical because La/SSB is essential for eukaryotic life and is required both for dividing and non-dividing postmitotic cells, which contribute to the development of normal tissues. 27 The La/SSB protein performs a versatile range of chaperone functions for many different RNA molecules and thus regulates both transcription and translation. [28][29][30][31][32] La/SSB is integral to the processing of various small non-coding RNAs including such precursor transcriptional products of RNA polymerase III as pre-tRNA and pre-5S rRNA molecules, precursor microRNA molecules (miRNAs) [32][33][34] and, by implication, probably also of DNA damage response miRNAs called Drosha-and Dicer-dependent small RNAs (DDRNAs). 35 Here, La/SSB protects nascent pre-tRNAs and pre-miRNAs from exonucleolytic degradation and stabilises or 'holds' the stem-loop structure of miRNAs to modify their levels of expression, and to promote miRNA-mediated cleavage of mRNA. [32][33][34][35] Via different mechanisms, La/SSB can stimulate translation of viral and cellular mRNA molecules that play important roles in viral replication, malignant processes and cellular stress responses. Although it was first shown for polio and hepatitis C viruses that La/SSB can act as an internal ribosome entry site (IRES) transactivating factor, or ITAF, and promote cap-independent translation of mRNA by IRES binding, La/SSB has been shown to function as an ITAF during cellular stress to promote cap-independent translation of MDM2, 17,18 XIAP,36 BiP/GRP78, 37 Laminin B1, 23,24 CCND1 14 and NRF2. 25 In other cases, La/SSB destabilises a stem-loop structure, which embeds a translation start site and promotes ribosomal scanning, and thus stimulates the translation of mRNA for the pro-survival gene, Bcl2. 16 Although La/SSB is predominantly located in the cell nucleus, it can move from the nucleus to the cytoplasm particularly after infection, 38 cellular stress, 39,40 and during cell death when caspase-mediated cleavage of the 3 kDa C-terminal nuclear localisation signal results in cytoplasmic translocation of La/SSB. 41,42 Among the putative oncogenic roles of La/SSB overexpression is resistance to cisplatin, which has been demonstrated in cell lines of the aerodigestive tract cancer, HNSCC, and in which knock-down of La/SSB was shown to sensitise the cells to cisplatin. 16 In an earlier study, reducing La/SSB expression was shown to sensitise chronic myeloid leukaemic cells to chemotherapy. 16,17 Together, these data suggest that La/SSB may be involved either in protection from DNA damage or repair of treatment-induced DNA damage. Therefore, we made an initial series of experimental observations to address the gap in our understanding of the conditions and context for binding of the chDAB4 mAb to tumour cells dying after DNAdamaging treatment and to explore the potential involvement of La/SSB in the DNA repair response to DNA-damaging treatment in lung cancer cells. Numbers of γ-H2AX foci and Rad51 foci were used to evaluate the extent of DNA damage caused by DSB overall and the subset of DSB potentially reparable by the homologous recombination DNA repair mechanism, respectively. In this study, we performed a more detailed analysis of the interaction of La/SSB with radiation-induced DSB in three lung cancer lines to identify if La/SSB is recruited to DNA DSB using sensitive imaging techniques and co-immunoprecipitation.
Previously, we observed that, in response to DNA-damaging stimuli such as ionising radiation, the levels of La/SSB expression in tumour cells increased before plasma cell membrane integrity was lost. 12,21 Hence, immunocytological observations of La/SSB protein interactions in the current study were made after fixation and permeabilisation of the cancer cells.
2 | RESULTS 2.1 | X-radiation induces DNA damage including DSB in lung cancer cells ɣ-H2AX and Rad51 were used as biomarkers of DSB. Rad51 is a key protein marker of error-free repair of DNA by homologous recombination and helps to maintain genomic integrity and stability. An increase in the number and size of nuclear Rad51 foci is a hallmark of the early cellular response to DNA damage. We first examined the DNA damage response to escalating doses of ionising radiation in the human lung cancer lines, A549 and H460, and the murine Lewis Lung (LL2) carcinoma cell line. Cells were exposed to increasing radiation dose with 0, 1.25, 2.5 or 5 Gy and DNA damage was assessed after 4 h using the DNA damage markers ɣ-H2AX and Rad51. A549 cells were the most radio-resistant, with lower numbers of residual ɣ-H2AX ( Figure 1A, top row) and Rad51 foci ( Figure 1A, bottom row) with increasing radiation dose at 4 h after radiation. H460 cells were more sensitive, with the number of ɣ-H2AX ( Figure 1B

| Immunofluorescence analysis of La/SSB expression and radiation-induced DSB formation
To investigate whether La/SSB associated with DNA damage markers, we examined the expression of La/SSB and ɣ-H2AX by fluorescence microscopy in untreated and irradiated lung cancer cells. Although the fluorescence signals indicated co-localisation of La/SSB with radiation-induced ɣ-H2AX foci (Figure 3), it is difficult to confirm if these proteins interact directly because of the dominant fluorescence signal emanating from the abundant and ubiquitous nuclear La/SSB protein. To elucidate further whether these proteins co-localised, we generated relative intensity plots of La/SSB and ɣ-H2AX staining using regions of interest in cells that appeared to co-express both La/SSB and ɣ-H2AX (represented by the line in the merged image in Figure 3). From this analysis, we identified varying intensities of La/SSB throughout the cell, and in most cases La/SSB expression increased at the same sites where ɣ-H2AX foci were present, thus suggesting an accumulation of La/SSB specifically at the DSB site.  F I G U R E 4 Proximity ligation assay analysis using antibodies specific for La/SSB and γ-H2AX in lung cancer cells treated with radiation. A549, H460 and LL2 carcinoma cells were untreated or irradiated with varying doses of radiation. Four hours later, cells were stained with La/SSB-and γ-H2AX-specific antibodies, which had been labelled with Duolink in situ probe maker and developed using Duolink In Situ Detection reagents. Shown are the number of PLA foci per nucleus with significant differences compared to untreated cells. PLA foci of at least 50 nuclei were counted. Each point represents the count of an individual nucleus in the graphs. The cells were imaged using a 63 Â oil immersion objective with a 3 Â zoom factor. Scale bar, 5 μm and ɣ-H2AX (Figures S3 and S4).
To further confirm a physical interaction between the La/SSB and ɣ-H2AX proteins, the La/SSB protein was pulled down from whole cell lysates using chDAB4-protein A Sepharose beads and the resulting protein was probed for ɣ-H2AX by Western blotting. In our hands, we could not pull down La/SSB from A549 cells, so we pulled down ɣ-H2AX and probed this protein sample for La/SSB with chDAB4. Radiation increased ɣ-H2AX protein in all cell lines, particularly at 30 min after irradiation ( Figure S1B), and when immunoprecipitation was performed, it was confirmed that La/SSB and ɣ-H2AX were bound together ( Figure 5B) with full-length blots and gels presented in Figure S2C. In contrast, no signal was obtained using the isotype antibody-bound Sepharose beads.

| DISCUSSION
The DNA damage response (DDR) comprises a highly redundant system for the crucial protective task of rapidly repairing DNA damage, particularly the DSB, which, unless it is repaired, will not permit continued survival of the cell. Although components of the DDR system are often impaired during carcinogenesis, mutational and nonmutational mechanisms in cancer cells may improve the control and efficiency of this system during DNA damaging treatment and thus contribute to treatment resistance.
Given that DNA damage may happen as rapidly as electron-transfer, the transcripts involved in the DDR are expressed before the DNA repair process begins, and dynamic and intricate regulation of transcript stability allows cells to react promptly to the damage and maintain genomic integrity. The DDR involves at least hundreds of RNA molecules and proteins including mRNA, non-coding RNA molecules and RNA-binding proteins (RBP). In response to DSB induced by ionising radiation, activated ATM phosphorylates the histone variant H2AX on Ser139 to form γ-H2AX. This key step in signal amplification enables recruitment of additional DDR mediator proteins, 43 which in turn recruit more ATM-containing complexes, thus establishing a positive feedback loop. 35 A maximum number of γ-H2AX foci form 10-30 min after irradiation.
The stoichiometry suggests that hundreds to several thousand γ-H2AX molecules surround each DSB 44 with the positive feedback signalling F I G U R E 5 Temporal proximity ligation assay analysis and co-immunoprecipitation of La/SSB protein with γ-H2AX after X-irradiation in lung cancer cell lines. (A) A549, H460 and LL2 cells were irradiated with 5 Gy and 0.5, 4 or 8 h later cells were stained with La/SSB-and γ-H2AXspecific antibodies, which had been labelled with Duolink in situ probemaker and developed using Duolink In Situ Detection reagents. Shown are the number of PLA foci per nucleus with significant differences compared to untreated cells. PLA foci of at least 50 nuclei were evaluated and each point represents the count of an individual nucleus. The cells were imaged using a 63 Â oil immersion objective with a 4 Â zoom factor. Scale bar, 5 μm. (B) Protein lysates from untreated or treated cells were co-immunoprecipitated using protein A Sepharose beads bound with either DAB4 (H460 and LL2 cells) or γ-H2AX antibody (A549 cells) or protein A Sepharose beads bound with isotype control antibody was used as a control. Immunoprecipitated (IP) samples were analysed by Western blot using biotin-γ-H2AX antibody (for H460 and LL2 cells) or chDAB4 (A549 cells) enabling γ-H2AX to spread for hundreds of kilobases beyond the DSB, permitting cytological detection of DDR foci. 35 Furthermore, γ-H2AX facilitates recruitment of DNA repair protein complexes that include RAD51, which is involved in homologous recombination repair of DSB. 35 As a RBP, La/SSB is engaged in most steps of miRNA processing including indirect RNA-mediated interactions with Drosha and Dicer, which are catalytic engines of miRNA biogenesis and which together with γ-H2AX are essential for secondary recruitment of DDR factors and thus the amplification of DDR signalling. 45,46 In addition to its presumed localisation at DDR foci in the nucleus at the time of DNA damage, La/SSB can also be found in the nucleus and cytoplasm bound to the 5 0 UTR of mRNAs and to the stem-loop structures of pre-miRNAs. [32][33][34]47 Interestingly, La/SSB is one protein found to associate with γ-H2AX in unirradiated cells 48 and in cisplatin-treated cancer cells. 12 La/SSB is also a calmodulinbinding protein 49 and calmodulin is upregulated after radiation exposure and is involved in the γ-H2AX-mediated DNA repair pathways. 50,51 It is becoming apparent that overexpression of La/SSB in cancer with DNA-damaging agents, we examined in more detail whether La/SSB was present at the DNA DSB site. 12 In the three lung cancer lines analysed herein, radiation induced a rapid, dose-dependent formation of ɣ-H2AX and Rad51 foci. Indeed, our data indicating co-localisation of La/SSB with γ-H2AX suggest that La/SSB is present at DDR foci as early as even 30 min after radiation-induced DNA damage. Since La/SSB is a RBP involved in nuclear processing of miRNA and, by implication, probably also of DDRNAs, RNA-bound La/SSB may already be present in abundance at the instant that DNA damage occurs as well as be rapidly induced as part of the DDR.
The repair kinetics differed between the two human lung cancer lines, with a reduction in γ-H2AX and Rad51 foci at 4 h after irradiation in A549 cells compared to H460 cells, suggesting faster repair of DSB in A549 cells compared to H460 cells. Similar to our results, it has been shown by others that A549 exhibit fewer γ-H2AX and Rad51 foci after irradiation than H460 cells. 52 Furthermore, Sak et al. showed elevated Rad51 foci in γ-irradiated H460 cells compared to A549 and that H460 cells had a higher fraction of residual Rad51 foci, which is predictive of radiosensitivity. 53 Indeed, the A549 cell line is more radio-resistant than the H460 cell line and shows reduced radiation-induced apoptosis, which could be explained by faster or more efficient repair kinetics.
Although in a separate study, Yu et al. did not find any differences in the repair kinetics between H460 and A549 cells irradiated with 2 Gy, they did note that radiation increased autophagy and senescence in H460 cells compared to A549 cells. 54 Compared to H460 cells, A549 cells have a higher expression of the nuclear factor erythroid-2 related factor 2 (NRF2). 55 NRF2 is a transcription factor that regulates antioxidant genes and its activation increases repair of radiation-induced DNA damage. 56 Given that La/SSB can increase NRF2 protein translation from oxidative stress, 25 radiation-induced expression of La/SSB may contribute to the radio-resistance of A549 cells.
In keeping with our previous findings, there was co-localisation of La/SSB at DNA DSB by immunofluorescence imaging techniques, and this interaction was confirmed both by PLA and co-immunoprecipitation of La/SSB and γ-H2AX. We found that the proportion of PLA foci was inversely proportional to the number of γ-H2AX foci in the treated human lung cancer lines. That is, although A549 cells had, on average, fewer γ-H2AX foci after irradiation compared to H460, they did have more PLA foci. Given that γ-H2AX foci were resolved more quickly in A549 cells than in H460 cells after X-radiation, we postulate that interactions of La/SSB protein with RNA molecules or other proteins at the DNA DSB site, which are marked by the PLA foci, contribute to faster DNA repair. Although murine LL2 cells, which have the highest PLA signal number compared to human H460 cells, may have a different mechanism to account for their slower DNA repair kinetics, which is also observed in H460 cells.
Finally, a specific relationship to DNA-damaging treatment of the interaction between La/SSB and γ-H2AX was confirmed by using cisplatin and mitomycin C as cytotoxic inducers of DNA damage including DSB or vinorelbine, which is a microtubulin-binding agent. In contrast to the time-dependent appearance of PLA foci after treatment with the DNA-damaging drugs, few PLA foci were observed after treatment with vinorelbine irrespective of the period of observation. In this respect, foci of γ-H2AX have been observed as the result of apoptotic endonuclease-mediated chromatin cleavage and before apoptotic cell death is evident. 57 Of course, it must be recognised that this study has its limitations.
For example, tumour microenvironmental effects, which are not investigated here, increase the biological complexity of the DNA damage response. 58,59 And it is known that ionising radiation and radiomimetic drugs such as platinating agents can produce clustered DNA damage, which comprises complex arrangements of single-strand damage and which may or may not include DSB. 60,61 Investigation of specific mechanisms of DNA repair is beyond the scope of this study.
In summary, we found that La/SSB localised at the DSB site after ionising radiation or DNA-damaging cytotoxic drugs. We hypothesise that co-localised staining of La/SSB with γ-H2AX, which we identified using PLA, represents a very minor subset of all possible La/SSB molecules but only the molecules that are present in association with DSB. Although we do not know why La/SSB might associate with DSB, the fact of its abundance and ubiquity as a multifunctional RNA-binding molecule may mean that La/SSB is present as an 'innocent bystander'.
We hypothesise that the known role of La/SSB in binding of miRNA molecules together with the emerging role described for the miRNA subset of DDRNAs may account for the presence of La/SSB at DSB in close proximity to γ-H2AX. Alternatively, La/SSB may play an active role in the DNA-damage response by facilitating DNA repair, a proposition we wish to test in future studies. Although further studies are required to investigate the mechanisms underlying these hypotheses, the results of this study do posit an explanation for why significantly higher binding of the chDAB4 to dead tumour cells is found after DNA-damaging anti-cancer treatments such as ionising radiation and platinating drugs.  No. ab6721, Abcam) for 1 h at room temperature. The membrane was imaged using a Bio-rad ChemiDoc MP system and analysed using Imag-eLab™ software. Densitometry was performed using software ImageJ software (National Institutes of Health, Bethesda MD).

| Cell cultures
For co-immunoprecipitation, protein lysates were collected as above, primary antibody (murine DAB4 or ɣ-H2AX) added and incubated with rotation at 4 C for 1 h. Washed protein G beads (Cat.
No. 10-1242, Thermo Fisher Scientific) were added into the antibodylysate mixture and incubated overnight at 4 C with rotation. The beads were collected by centrifugation, washed with RIPA buffer and the bound protein released by heating at 95 C for 5 min. Samples were analysed by SDS-PAGE as described above, with 30 μg of protein lysate loaded per well. The La/SSB protein was detected using 5 μg/mL chDAB4 followed by 4 μg/mL anti-human IgG-HRP (Cat. No. ab99759, Abcam) and ɣ-H2AX detected using 1 μg/mL biotinylated mouse anti-phospho-H2AX (ser139) mAb (Cat.

| Statistical analysis
Statistical analyses were performed using GraphPad Prism (v7.0) software. Data were tested for normality using the D'Agostino's K-squared test. For normally distributed data, an unpaired twotailed t-test was used to compare two groups, and one-way ANOVA was used to compare three or more groups. For data that were not normally distributed, the Mann-Whitney test was used to compare two groups and the Kruskal-Wallis test to compare three or more groups. Data are shown as mean ± standard error of the mean, and p-values are shown. Unless otherwise stated, significance values are when compared to untreated cells and *p < .05, **p < .01 and ***p < .001.

CONFLICT OF INTEREST
Michael P. Brown is co-inventor on APOMAB patents owned by AusHealth Pty Ltd. and no competing interests exist for other authors.

AUTHOR CONTRIBUTIONS
All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analy-

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICAL STATEMENT
No ethical statement is required because this work was done using human cancer cell lines rather than primary human cancer cells, and no animal experiments were performed.