NUTM1 with a chromatin-binding partner, typically the bromodomain-containing protein BRD4. However, little is known about other genetic aberrations in this disease. In this study, we used a unique panel of cell lines to describe the molecular-genetic features of NMC. Next-generation sequencing identified a recurring high-impact mutation in the DNA-helicase gene RECQL5 in 75% of lines studied, and biological signals from mutation-signature and network analyses consistent with a general failure in DNA-repair. A high-throughput drug screen confirmed that microtubule inhibitors, topoisomerase inhibitors and anthracyclines are highly cytotoxic in the majority of NMC lines, and that cell lines expressing the BRD4-NUTM1 (exon11:exon2) variant are an order of magnitude more responsive to bromodomain inhibitors (iBETs) on average than those with other BRD4-NUTM1 translocation variants. We also identified a highly significant correlation between iBET and aurora kinase inhibitor efficacy in this study. Integration of exome sequencing, transcriptome, and drug sensitivity profiles suggested that aberrant activity of the nuclear receptor co-activator NCOA3 may correlate with poor response to iBETs. In conclusion, our data emphasize the heterogeneity of NMC and highlights genetic aberrations that could be explored to improve therapeutic strategies. The novel finding of a recurring RECQL5 mutation, together with recent reports of chromoplexy in this disease, suggests that DNA-repair pathways are likely to play a central role in NMC tumorigenesis.NUT midline carcinoma (NMC) is a rare and aggressive cancer, with survival typically less than seven months, that can arise in people of any age. Genetically, NMC is defined by the chromosomal fusion of  While little is known about the cellular role of NUTM1, the NUTM1-partner genes  are recognized as master regulators of chromatin structure and function. Recent studies have shown that the NUTM1 component of NMC fusion proteins can recruit histone acetyltransferases, such as p300 and CREB-binding protein [MYC and TP63 [NUT midline carcinoma (NMC), also known as NUT carcinoma, is an invariably fatal malignancy with an average survival time of less than 7 months . The tumsurvival . The dis protein , whilst  protein –8. Howevand TP63 .BRD4 in cancer has led to the development of a new generation of anti-cancer compounds that specifically target the BET  family of proteins, of which BRD3 and BRD4 are key members [et al. have shown that a key component of the oncogenic mechanism in the majority, if not all NMC tumors, is the formation of an iBET-sensitive complex involving NSD3, BRD4 and NUTM1 [Recognition of the importance of  members –13. Impond NUTM1 . As a rend NUTM1 .MYC; [Despite this preliminary report and a wealth of encouraging laboratory data, several pre-clinical studies and clinical trials have indicated that the therapeutic benefit of iBETs may be limited by toxicity at higher doses, and by the acquisition of resistance –20. Two MYC;  whilst iWe previously demonstrated that the efficacy of BET-inhibition in NMC might vary in regard to either the precise cell of origin of each tumor, or the specific chromosomal translocation involved . In the BRD4-NUTM1 and BRD3-NUTM1 fusion variants, with the majority of the lines having either a BRD4-NUTM1 ex11:ex2, or a BRD4-NUTM1 ex15:ex2 breakpoint (NUTM1) and verified that this line indeed carries an NMC breakpoint. This finding was independently confirmed in another publication whilst conducting this study [NUTM1 in an undifferentiated carcinoma is diagnostic for NMC, and (ii) it reinforces the fact that NMC has historically been under-diagnosed, highlighting the potential for further discovery of samples in bio-banks around the world.The rarity of NMC significantly limits the availability of tumor material, thus tumor-derived cell lines provide an invaluable resource for further research. In this study, we compared the drug response profiles and the genetic features of 12 NMC cell lines  and poor responder (n = 7) groups were comprised of cell lines from all three phenotypes , only NMC lines (n = 4) were represented in the sensitive group . When further segregated based on NMC gene-fusion subtype  or non-NMC carcinomas (p < 0.0001). It is notable that the one cell line carrying a BRD3-NUTM1 translocation (10326) was also highly sensitive to iBET treatment. A similar response pattern was observed for the iAURKs, with analysis confirming a highly significant correlation between iAURK and iBET sensitivities . This observation is consistent with the findings of a recent report describing a relationship between iBET treatment and the suppression of AURKs [Subsequent unsupervised hierarchical clustering focusing solely on iBET responses across the cell line panel, identified three distinct groups of iBET sensitivity Figure , which we Figure , iBET trof AURKs . AlthougNUTM1 translocation in P896-FB was confirmed by both genomic PCR, and RT-PCR, and the karyotype was normal , which plays an important role in cellular differentiation. Finally, there were variants in genes involved in DNA-damage response and apoptosis , as well as control of chromosome-separation during cell division . The latter observation is of particular interest in the context of a disease for which the driving oncogenic feature is a chromosomal translocation event.Due to the scarcity of NMC, and its historical under-diagnosis, matched tumor-normal samples for next-generation sequencing analysis are rare. However, from a previously described NMC patient (P896)  we were s normal . We thuss normal . Althougs normal . SeveralAs a final comparison, we ran the same GATK pipeline to call variants from WES data obtained from 11 of the NMC cell lines, however very few of these P896 germline candidates (only 10 out of 179) were found to be present in any of these lines, and there were none that were present in more than one NMC cell line , arguingThe fibroblast line P896-FB was then used as a control to call somatic mutations in the primary NMC sample P896 using two different paired tumor-normal algorithms, MuTect analysis  which c had alreThe somatic mutation rate calculated for NMC cell lines ranged from 2.0–3.8 mutations/Mb Figure , whilst http://cancer.sanger.ac.uk/cosmic/signatures) [To gain a better understanding of the mutagenic processes that underpin such genetic alterations, we next assessed the summarized nucleotide transition/transversion (Ti/Tv) ratios for each of the samples. The Ti/Tv ratios varied from an average of 2.10 in the NMC cell lines (range: 1.58–2.54) to 2.8 in the P896 primary sample, values that are similar to the WES estimates of ~2.8 from the 1000 Genome Project Consortium , 46. Hownatures) , 48, 49.natures) , 50, 51.FAM104B, HYDIN, KIR2DL1, RECQL5, TTN and ZNF717) harbored variants in at least two-thirds of the NMC samples  to examine gene expression profiles across the cell line panel. Following RNA extraction and sequencing, we normalized RNA-Seq read counts to extract baseline gene expression estimates from NMC cell lines and the non-tumor line P896-FB , and looNUTM1 being absent in this control line in contrast to the positive expression seen in the NMC lines. Expression of the neuroblastoma tumor suppressor gene CASZ1 was also notably absent in P896-FB, in contrast to the NMC lines . Using these methods, we were unable to detect differences in RECQL5 splicing in lines carrying this variant, however it is important to remember that different splicing programs and factors may be brought to bear during differentiation, tumorigenesis, response to therapy, and other cellular contexts. As such, it is reasonable to suggest that differences in the transcriptional processing of RECQL5 in mutated lines may only become evident under different cellular conditions or stages of differentiation.We therefore restricted the analysis to focus on genes affected by high-confidence deleterious variants  were found to carry moderate impact variants in RPMI2650 and PER-624 (NCOA3 (20_46279815_GCAGCAGCA/-) had obvious biological relevance for NMC. NCOA3 is a nuclear receptor co-activator with histone acetyltransferase activity that recruits p300/CBP and CREB binding protein as part of a multi-subunit co-activation complex. The identified variant has previously been reported in a single study in breast cancer , and the deletion lies within a poly-glutamine (poly-Q) sequence in the carboxyl-terminal acetyl-transferase domain of the protein. Findings from Wong et al. suggest that a shorter poly-Q domain may increase the co-transactivation activity of NCOA3, potentially resulting in a more aggressive form of cancer [NCOA3 variant (RPMI2650 and PER-624), while the other two (TC797 and P896-CL) had significantly higher NCOA3 expression compared to all other samples. In other carcinomas, high expression of this gene has been shown to be associated with tumor progression, metastasis and chemoresistance [NCOA3 in a subset of NMC cell lines, caused by either a deletion in the poly-Q domain or via an increase expression, that contributes to the attenuation of iBET cytotoxicity.In an alternative approach to identify important genetic drivers of disease progression, we next investigated whether the attenuated response to iBET treatment that we observed in a subset of NMC cell lines  and the matching non-tumor fibroblast line P896-FB  were established from clinical biopsies of NMC patient P896 . The NMCDrug screening was conducted at the Children's Cancer Institute Drug Discovery Centre, Sydney, Australia, and assessed the toxicity of 23 compounds in 12 NMC cell lines, six non-NMC carcinoma lines and two non-cancer fibroblast lines. Compounds were obtained from the following sources: docetaxel, vincristine sulfate, methotrexate, vorinostat, gefitinib, erlotinib, alisertib/MLN8237, AMG-900, epothilone B, JQ1, OTX-015 ; pyrvinium pamoate, digoxin, barasertib/AZD1152 ; I-BET151, GSK-2801 ; flavopiridol ; PFI-1 ; gemcitabine, daunorubicin, topotecan, mitoxantrone, monensin . Cells in log-phase growth were seeded in 96-well assay-ready plates using a Multidrop-384  and incubated for 96 hours at 37°C in the presence of the indicated drug or vehicle (DMSO). Screening was performed with drug concentrations from 0.0025 μM to 10 μM , with IC50 values subsequently determined in two independent experiments. Response to drug treatment was determined by dispensing 10% (v/v) Alamar Blue reagent to assay plates using a Multidrop-384 (Thermo Scientific). Following a 6 hour incubation at 37°C metabolic activity was determined by measurement of fluorescence intensity  using an EnSpire plate reader . Percentage cell viability was calculated relative to positive and negative (vehicle only) controls. Data analysis included generation of dose response curves and calculation of IC50 values using the ActivityBase software suite . Unsupervised hierarchical clustering of cell lines and drugs based on mean IC50 values was performed using the software Genesis [Genomic DNA was isolated using the DNeasy Blood & Tissue Kit  from 11 NMC cell lines , as well as the primary sample P896 and the P896-FB fibroblast line grown from this patient. Whole exome 100bp paired-end sequencing (WES) was performed at AGRF  using the Agilent SureSelect QXT Human All Exon +UTRs v5 (75Mb) target capture kit according to manufacturer's protocols, with samples multiplexed on the Illumina HiSeq 2500 platform. For details of the bioinformatic pipelines used for variant detection and the analysis of mutational signatures, please refer to the Supplementary Materials.Total RNA was isolated using TRIzol  and the RNeasy Mini Kit  from 12 NMC cell lines  and the P896-FB fibroblast line according to the manufacturers’ protocols, with QC performed using the Bioanalyzer 2100 . Sequencing libraries were prepared with Illumina's TruSeq stranded polyA protocols and were processed on an Illumina HiSeq 2000 (100bp paired-end sequencing) at AGRF . For bioinformatic pipelines used for gene expression estimates and statistical analysis, please refer to the Supplementary Materials.The raw data (BAM files) for all exome and transcriptome analyses performed in this study can be accessed from the NCBI's Short Read Archive (SRA), under Project Accession PRJNA339503 (SRA Accession SRP083924).


CEP290  being the most frequently mutated gene. The most recurrent LCA-causing CEP290 mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of CEP290 transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in CEP290. We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat CEP290-associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with  CEP290) are the most frequent cause of LCA in some populations. The protein encoded by this gene is localized at the base of the connecting cilium of the photoreceptors, which is an essential structure to transport proteins from the inner segment, where they are synthesized, to the outer segment, where phototransduction takes place. [Leber congenital amaurosis  is the most severe subtype of inherited retinal disease (IRD), and is characterized by a congenital visual impairment, progressive degeneration of photoreceptor cells, sensory nystagmus, and the absence or significant reduction of electroretinographic responses in the first year of life . So far,s place. ,6. CEP29s place. ,7, is ins place. . As a cos place.  which haCEP290, c.2991+1655A>G, accounts for 15% of all LCA cases in some European and North-American populations [CEP290 mRNA transcripts contained the pseudoexon upon differentiation of patient-derived iPSCs to photoreceptor-like cells [Despite the existing high genetic heterogeneity in LCA, one particular mutation in ulations ,12,13. Tulations ,15. HoweCEP290, AONs have been demonstrated to restore splicing, protein levels and ciliation in patient-derived immortalized lymphoblastoid cells [CEP290 splicing in the retina of a transgenic humanized mouse model, without creating any damage in the retina [CEP290 splicing and the cellular phenotype in compound heterozygous cases, by using patient-derived fibroblast cells where only one CEP290 allele is represented by the deep-intronic c.2991+1655A>G mutation.The recurrence of this intronic mutation makes it an interesting target for genetic therapy. Over the last years, we and others have shown that antisense oligonucleotides (AONs) are a powerful therapeutic tool to treat IRDs ,18,19,20id cells , fibroblid cells ,17 and pid cells . In addiCEP290 mutations. In all six cases, at least one of the two alleles harboured the deep-intronic mutation c.2991+1655A>G. Three patients were homozygous for this variant (LCA1-LCA3), whereas three others (LCA4-LCA6) carried protein-truncating mutations on the other allele. In two patients the p.Gly1890* variant was reported as the second disease-causing allele (LCA4 and LCA6), while in the other (LCA5), the p.Lys1575* mutation was present. In addition, we also obtained a skin biopsy from one of the parents of the homozygous LCA-affected individual LCA3, thus heterozygously carrying the c.2991+1655A>G allele (HET1). All affected individuals were diagnosed with LCA soon after birth and were otherwise healthy. In adulthood, subject LCA6 was found to be infertile due to immotile spermatozoa. This might fit within the spectrum of CEP290-associated non-ocular features, although there is no strong evidence supporting that. A summary of the genetic and clinical information for each individual is shown in Six patients were previously diagnosed with LCA caused by biallelic CEP290 pre-mRNA in cell lines derived from patients harbouring the deep-intronic mutation in a homozygous manner [CEP290.Fibroblast cell lines were generated from skin biopsies of all six LCA patients (LCA1-LCA6) and the heterozygous carrier of the c.2991+1655A>G mutation (HET1). In addition, age and gender-matched control cell lines were used. Subsequently, we studied the effect of AON delivery at the RNA, protein and cellular level. Previously, we have identified an AON molecule that is able to efficiently restore s manner ,17. In tWe next assessed whether the splicing correction at the RNA level could be translated to an increase in the CEP290 protein levels. For that, an immunodetection of the protein by Western blot was performed. Given the amount of samples, it was not possible to have a direct comparison of all samples on the same gel, and thus we compared untreated versus AON-treated conditions for each cell line. A representative image of the CEP290 protein levels in all cell lines is illustrated in CEP290 is that a cellular phenotype is present. CEP290 is known to function in cilium transport and within the cell cycle, and previously, we and others have observed that in fibroblasts derived from individuals with CEP290 mutations, these cells show shorter cilia, less ciliation and sometimes even a slower growth speed [One characteristic of fibroblast cells carrying mutations in th speed ,16,17,22th speed ,16,17. ICEP290 pre-mRNA splicing, increased the CEP290 protein levels and restored a cellular defect in fibroblast cells homozygously carrying the c.2991+1655A>G mutation. These results have recently led to the initiation of a clinical trial , assessCEP290-homozygous cell line LCA3 stopped growing and therefore Western blot and ciliation experiments were impossible to perform. One of the reasons could be that when the skin biopsy was received, a contamination was suspected, and the cells were subjected to antifungal treatment with primocin. Any traces of contamination could not be detected afterwards, but this could have had an effect on their normal growth and/or morphology. Another possibility is that these cells carry other variants that may influence the normal cell cycle. In general, we observed that our cell lines were growing at different speeds. On average, the control cell lines, as well as the one derived from the carrier, had a normal speed. The homozygous cell lines, with the exception of the LCA3 case, grew moderately slower than controls. Two of the three cell lines derived from the compound heterozygous patients however grew noticeably slower than the rest of the cell lines. These differences could be related to the severity of the second CEP290 allele, which represents a protein-truncating mutation. Importantly, we and others have previously described that in c.2991+1655A>G homozygous fibroblast cells, the ratio between correct and aberrantly spliced CEP290 transcripts on average is ~1:1. This indicates that in a homozygous situation, around 50% of the transcripts are still correct, while in a compound heterozygous situation this is only 25%. Given that CEP290 has been related to cell division, this could explain the evident differences in growth that are observed [Our study design consisted of three groups of cell lines; the control group, the homozygous group and the compound heterozygous group, together with one carrier cell line. For RNA analysis, we were able to use all cell lines. However, after a very few passages, the observed ,24,25,26CEP290 pre-mRNA splicing, increasing the protein levels and correcting a ciliary defect [CEP290 cause recessive forms of several diseases, we hypothesized that correcting one allele should be enough to restore CEP290 levels and function in the cell. Therefore, we assessed fibroblast cells derived from patients carrying the deep-intronic variant together with a protein-truncating mutation on the second allele. In two cases, the p.Gly1890* variant (LCA4 and LCA6), and in a third one (LCA5), the p.Lys1575* variant were present. Previously, both these mutations were reported to be associated with more severe forms of CEP290-associated diseases, i.e., the p.Gly1890* variant with cerebello-ocular-renal syndrome (CORS) and Joubert Syndrome, whilst the p.Lys1575* variant with CORS, Senior-Løken syndrome and Meckel-Grüber syndrome-like , and compound heterozygous lines (LCA5 and LCA6). For LCA4 there was an apparent increase. This rise in CEP290 protein levels also resulted in an increased length of the cilium in all patient-derived cell lines, albeit not always statistically significant. In our previous study, cell lines LCA1 and LCA2 did show a statistically significant increase in ciliation and cilium length following AON treatment [The delivery of AONs to cells homozygously harbouring the c.2991+1655A>G mutation was shown to be effective in restoring y defect ,15,16,17n = 256 for untreated and n = 209 for the AON-treated), however this was taken into account when applying the statistical test. When evaluating the two replicates independently, one showed a statistically significant difference whereas the other did not. When both replicates were combined, these differences became more evident. All samples were treated simultaneously so potential technical problems or alterations due to the reagents could be discarded as this should have then also been observed in other samples. We included age- and gender matched control cell lines, used as lowest cell passages as possible, and tried to compensate the growth speed by seeding more or less cells of each cell line. Nevertheless, the variability between samples and groups was higher than expected.In our study, we also used a cell line of an unaffected heterozygous carrier of the c.2991+1655A>G mutation to assess whether any ciliation defect could be observed in these cells. As expected, the amount of pseudoexon was low, and levels of protein did not show an evident increase after treatment, nor a significant decrease compared to the control cells. Surprisingly, although these cells showed normal levels of ciliation, a statistically significant decrease of the cilium length was detected upon AON administration. Even though both groups followed a Gaussian distribution with a relatively similar cilium length average ± SD of 4.08 µm ± 1.05 for the untreated and 3.88 µm ± 1.09 for the AON-treated group, the medians differed between groups . One would assume that one reason could be the amount of cilia counted . In thi03140969 ,28,29. I03140969 ,31. How CEP290 pre-mRNA splicing, increased the CEP290 protein levels and restored a cellular defect in fibroblast cells homozygously carrying the c.2991+1655A>G mutation. Here, we assessed whether the correction of a single allele can also have a potential therapeutic effect. Overall, our results suggest that correcting one allele might be enough to minimize the effects of the pathology, although the severity of the second variant or the presence of other variants in ciliary genes needs to be taken into account.In summary, we previously identified an AON molecule that corrected n = 2). The AON efficacy was assessed at RNA level by RT-PCR, at protein level by Western blot and at cellular level by immunocytochemistry (ICC).This study was performed using a total of 10 fibroblast cell lines. Groups were divided as follows: three control lines derived from healthy individuals; three c.2991+1655A>G homozygous lines derived from LCA patients; three compound heterozygous lines from LCA patients carrying the c.2991+1655A>G in one allele and a stop mutation in trans (two of them: p.Gly1890* and another p.Lys1575*) and one carrier individual  presenting the deep-intronic variant on one allele. For all experiments all cell lines were processed at the same time and all experiments were performed in duplicate . Written informed consent was obtained from all participating individuals.CEP290-associated LCA and healthy or carrier controls were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 20% fetal calf serum (FCS), 1% penicillin-streptomycin and 1% sodium pyruvate at 37 °C and 5% CO2. For each analysis, cells were seeded in the corresponding plates. The next day, cells were transfected with AON  using FuGENE® HD Transfection Reagent  according to the manufacturer’s protocol.Fibroblast cell lines derived from individuals with 2O. PCR was performed with 10 µM of each primer pair, 2 µM of dNTPs, 1.5 mM MgCl2, 10% Q-solution , 1 U of Taq polymerase  and 5 µL of diluted cDNA in a total reaction of 25 µL using the following PCR conditions: 94 °C for 2 min, followed by 35 cycles of 20 s at 94 °C, 20 s at 58 °C and 35 s at 72 °C, with a final extension step of 2 min at 72 °C. Amplicons were resolved in a 2% agarose gel. Actin (ACTB) was used as loading control. For CEP290 the following primers were used: forward 5′-TGCTAAGTACAGGGACATCTTGC-3′ and reverse 5′-AGACTCCACTTGTTCTTTTAAGGAG-3′; for ACTB: forward 5′-ACTGGGACGACATGGAGAAG-3′ and reverse 5′-TCTCAGCTGTGGTGGTGAAG-3′.Cells were seeded and grown in 12-well plates and transfected with or without AON. Forty-eight-hour post-transfection, cells were harvested and subjected to RNA isolation using Nucleospin RNA isolation kit  following the manufacturer’s protocol. One microgram of RNA was used for cDNA synthesis using iScript cDNA synthesis kit . Subsequently, cDNA was diluted by adding 50 µL of HCells were grown in 10-cm dishes and transfected with or without 0.1 µM of AON. Forty-eight hours post-transfection, cells were serum-starved for another 48 h. Subsequently, cells were harvested and homogenized in 130 µL of RIPA buffer . Total protein was quantified using the BCA kit  as previously described ,17. Briet-test.Cells were grown on coverslips in 12-well plates and transfected with or without 0.1 µM of AON. Forty-eight hours post-transfection, cells were serum starved for another 48 h. Subsequently, cells were rinsed in 1× PBS, fixed in 2% paraformaldehyde for 20 min at RT, permeabilized in PBS supplemented with 1% Triton X for 5 min at RT and blocked for 30 min in 2% bovine serum albumin in 1× PBS at RT. For the immunostaining, cells were incubated 60 min in blocking solution containing a 1:1000 dilution of an anti-Acetylated α-Tubulin mouse monoclonal antibody  at RT. Cells were washed for 3 × 5 min in 1× PBS, incubated for 45 min with the corresponding secondary antibody  diluted in blocking solution. Cells were washed 3 × 5 min in 1× PBS and rinsed in water. Finally, coverslips were mounted in Vectashield with DAPI and imaged on a Zeiss Axio Imager Z1 Fluorescense microscope . For each condition, a minimum of 110 ciliated cells were analysed for cilium measurements using Fiji software . Samples


At present, advanced stage human Papillomavirus (HPV) negative and positive head and neck squamous cell carcinoma (HNSCC) are treated by intense multimodal therapy that includes radiochemotherapy, which are associated with relevant side effects. Patients with HPV positive tumors possess a far better prognosis than those with HPV negative cancers. Therefore, new therapeutic strategies are needed to improve the outcome especially of the latter one as well as quality of life for all HNSCC patients. Here we tested whether roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), which hereby also blocks homologous recombination (HR), can be used to enhance the radiation sensitivity of HNSCC cell lines.In all five HPV negative and HPV positive cell lines tested, roscovitine caused inhibition of CDK1 and 2. Surprisingly, all HPV positive cell lines were found to be defective in HR. In contrast, HPV negative strains demonstrated efficient HR, which was completely suppressed by roscovitine. In line with this, for HPV negative but not for HPV positive cell lines, treatment with roscovitine resulted in a pronounced enhancement of the radiation-induced G2 arrest as well as a significant increase in radiosensitivity. Due to a defect in HR, all HPV positive cell lines were efficiently radiosensitized by the PARP-1 inhibitor olaparib. In contrast, in HPV negative cell lines a significant radiosensitization by olaparib was only achieved when combined with roscovitine. Two main etiologies of head and neck squamous cell carcinoma (HNSCC) are recognized that reveal distinct biological characteristics. One is caused by infection with human Papillomavirus (HPV pos.), the other is mainly driven by noxa, such as tobacco and alcohol (HPV neg.) . In totaToday, both HNSCC entities are treated by a simultaneous cisplatin-based radiochemotherapy as a definitive concept or in the adjuvant setting after radical surgery, if tumors are in an advanced stage . For HPVMany different approaches were initiated already to implement new therapeutic strategies that specifically target HNSCC . HoweverIn order to achieve a tumor-specific effect, new treatments have to exploit tumor-specific defects, allowing for an individualized therapy. In this context cell-cycle regulation, which is accomplished by a complex network of different cyclin-dependent kinases (CDK) and respective cyclins, is considered to be of great relevance, because tumors often show defects in this process . As a coin vitro and in vivo [Roscovitine was one of the first CDK inhibitors targeting mainly CDK1 and CDK2 . The cyt in vivo , 10 incl in vivo . A recen in vivo . In cont in vivo . Two pha in vivo , 14.in vitro can modulate the response of classical chemotherapeutics [in vitro for breast cancer [Besides its activity as single agent, roscovitine apeutics  as well apeutics . An incrt cancer , nasophat cancer , as wellt cancer , 18. In We now tested, whether roscovitine may also be used to enhance the radiosensitivity of HPV neg. and pos. HNSCC cell lines. It is well known that the cellular radiosensitivity is primarily determined by the DSB repair efficiency . RoscoviIt was previously shown by us and others, that HPV pos. cell lines on average possess a higher radiosensitivity than HPV neg. cell strains –26. ThisWe describe here that roscovitine can be used to down-regulate HR in HPV neg. cell lines. Whereas roscovitine did not affect HPV pos. cells because HR was already impaired in these cell lines. In line with this, an increase in radiosensitivity by roscovitine was only achieved for HPV neg. but not for HPV pos. cell lines. The radiosensitivity of HPV neg. cells was further enhanced, when roscovitine was combined with the PARP1 inhibitor olaparib. In contrast, radiosensitivity of HPV pos. cell lines was only enhanced when pretreated with olaparib alone. The changes in radiosensitivity were strongly correlated with the respective variation in DSB repair capacity. These data confirm that the inhibition of DSB repair is an optimal tool to enhance the cellular radiosensitivity for both HPV neg. and pos. HNSCC cell lines. However, we also show that different strategies of radiosensitization are necessary to address the special characteristics of HPV neg. and pos. cell lines, respectively.CDKs are known for their essential role in cell-cycle regulation and proliferation . TherefoFigure Next, we tested the effect of ionizing radiation and roscovitine on the activity of CDK1 and CDK2, which is considered to be its primary target . The CDKFigure Besides their role in cell-cycle regulation, CDK1 and CDK2 are known to be relevant for the repair of DNA DSBs by HR . The quaTo confirm whether this is a general phenomenon in HNSCC cell lines, RAD51-foci formation was additionally measured in the other four HPV neg. and four pos. cell lines 4 and 10 h post IR Figure . In lineAn impaired HR is generally known to result in a pronounced G2 arrest when cells have to deal with un-repaired DNA-damage . TherefoOverall these data demonstrate that roscovitine specifically enhances the radiation-induced G2 arrest in HPV neg. cell lines resulting in an amount similar to the one observed in HPV pos. cell lines after irradiation alone. Since 10 μM roscovitine alone did not affect cell cycle progression, the increase in G2 arrest can be considered to result from the strong inhibition of HR achieved by roscovitine in HPV neg. cell lines as described above.We investigated in how far the effect of roscovitine on HR and cell-cycle distribution finally results in an altered number of unrepaired DSBs as can be analyzed by the detection of γH2AX foci. To this end, the number of residual γH2AX foci was measured 24 h after irradiation with 2 Gy using the HPV neg. cell line UM-SCC-3 and the two HPV pos. cell lines UD-SCC-2 and UM-SCC-47 Figure . In UM-SWe additionally analyzed the effect of the PARP-inhibitor olaparib in combination with roscovitine on radiation dependent γH2AX Foci. This inhibitor is known to depress the repair of DNA single-strand breaks (SSBs) . TherefoFinally, we investigated whether the different effects on DSB repair observed for HPV neg. and pos. cell lines treated either with roscovitine or olaparib alone or in combination prior to irradiation will also translate into different effects on cellular survival as determined by colony formation assay Figure . Both, tSimilar data were obtained when colony formation was measured in the other HPV neg. and pos. cell lines using a single dose of 4 Gy Figure . TreatmeAs shown for UD-SCC-2 Figure . SurprisThese data correspond well with our findings on residual γH2AX foci Figure , as alsoThe aim of this study was to test, whether the radiosensitivity of HPV neg. and HPV pos. HNSCC cell lines can be enhanced by the CDK inhibitor roscovitine.The study was performed with HNSCC cell lines previously described to carry the typical features of HPV neg. and pos. HNSCC tumors. For HPV neg. cell lines, these are a low expression of p16, mutated p53 and a low radiosensitivity, while for HPV pos. HNSCC cell lines a high expression of p16, wild-type p53 and an enhanced radiosensitivity are characteristic attributes –26. PrevRoscovitine is one of the first generation CDK inhibitors targeting a rather broad spectrum of CDKs via direct binding to the ATP site and with the highest impact on CDK1 and 2. Roscovitine alone was found to cause a slight increase of pCDK1 at T161 and pCDK2 at T160 Figure , which wet al. [An antiproliferative and cytotoxic effect of roscovitine was already observed in several other tumor models –10, whicet al. , studyinIt was previously reported that DSB repair is generally less effective in HPV pos. cell lines when compared to HPV neg. cell strains , 34, 35.The underlying molecular mechanisms causing a defect in HR in HPV pos. HNSCC cell lines are not yet fully understood. It was reported that RAD51 is over-expressed in HPV pos. cell lines due to the effect of oncoprotein E7 , but thaCDK1 and 2 are also known to be involved in HR. CDKs activate proteins, such as Exo1, BRCA1/2 and CtIp to promote early steps in HR that are necessary to induce RAD51 foci –23. As aIn line with these findings we also observed for HPV neg. cell lines that the radiosensitivity as determined by colony formation assay is clearly enhanced, when cells were pretreated by roscovitine, whereas for HPV pos. cell lines no such radiosensitization occurred Figure . Other aA defective HR is generally considered to be an ideal target to achieve a pronounced radiosensitization via the inhibition of PARP1 –44. ThisThe data presented here indicate, that this extended concept of synthetic lethality should also be applied to HPV pos. and neg. HNSCC tumors. It is confirmed here that the radiosensitivity of HPV pos. HNSCC cell lines can be enhanced by olaparib as already demonstrated by Güster et al.  and it iet al. [The different effects of roscovitine and/or olaparib on HR were found to result in clear differences in residual DSBs finally causing the respective effects measured for the cellular radiosensitivity as indicated by the excellent correlation of these two endpoints in the three cell lines tested . This waet al.  for someOverall, our data suggest that the extended concept of synthetic lethality using PARPi and ionizing radiation in HR deficient tumors appears to have a great potential for HNSCC. Due to this concept, HPV pos. tumors with a defective HR should be treated by IR in combination with olaparib alone, while for HPV neg. tumors this combination should be complemented by roscovitine to inhibit HR and thus obtain pharmacologically induced synthetic lethality.et al. [The concept of synthetic lethality always requires a stratification of tumors into HR proficient and deficient ones. However, so far a general and robust marker to identify such a defect in tumor samples is still missing. The data provided here Figure  and espeet al.  indicateIn summary, our data indicate that the response rates of HPV pos. HNSCC tumors might be further enhanced when combining IR with olaparib and, even more important, that the outcome of patients with HPV neg. tumors might be improved by adding roscovitine to this combination.The five HPV neg.  and the five HPV pos.  HNSCC cell lines used were described previously by us , except Irradiation was performed at room temperature using the X-RAD 320 iX X-ray tube . For inhibition of CDKs, roscovitine  was added in a final concentration of 10 μM, if not mentioned otherwise. PARP-1 was inhibited by olaparib  in a final concentration of 1 μM. If not stated otherwise, roscovitine was added 24 h and olaparib 2 h prior to irradiation and removed 24 h later. An equal amount of DMSO was used as solvent control.4 cells were seeded in 3.5 cm petri dishes and counted twice daily for 7 days. Roscovitine was added in increasing doses  directly while seeding cells. The number of cells was assed manually using a Neubauer chamber and cell number was calculated relative to the surface area of the petri dish. Dead cells were excluded by trypan blue staining and analyzed separately.For cell proliferation analysis, 8×10Cells were seeded in hexaplicates in 6-well plates in various cell numbers, depending on cell line. Roscovitine was added directly to cells while seeding and irradiation was performed 24 h later. In case of PARP-1 inhibition, cells were treated with olaparib 2 h before irradiation. All inhibitors were washed out 24 h after irradiation by a medium change using conditioned medium . In case of UD-SCC-1, 50% AmnioGrow Plus medium (Cytogen) was added to conditioned medium to facilitate colony formation. For colony formation cells were incubated for 9 to 21 days depending on cell line and treatment. Because treatment with inhibitors or irradiation delayed colony formation, treated samples were allowed to grow for extended periods of time and fixed when colonies reached equal size to control samples. After fixation and staining  colonies of at least 50 cells were counted manually and survival was calculated as previously described .Cells were grown on glass cover slips and incubated with roscovitine 24 h after seeding. After incubation for 24 h cells were irradiated with a dose of 2 Gy. For analysis of RAD51 foci, cells were fixed and stained at various time points (2-24 h) after irradiation with 4% paraform-aldehyde/PBS for 10 min and stored at 4°C over night. For evaluation of γH2AX foci the fixation was performed 24 h after irradiation. Fixed cells were permeabilized with 0.2% Triton X-100, 1% BSA/PBS for 10 min and blocked in 3% BSA/PBS for 1 h. Primary antibody incubation was done for 1 h at room temperature using the following antibodies: mouse monoclonal anti-phospho-S139-H2AX antibody , mouse monoclonal anti-RAD51 antibody  and rabbit polyclonal anti-CenpF antibody . After washing three times with 0.5% Tween20/PBS for 10 min, the cells were incubated for 1 h with secondary anti-mouse Alexa-fluor594  and anti-rabbit Alexa-fluor488 . Cells were again washed three times and mounted in ProLong Gold antifade reagent (Invitrogen) including DAPI for staining of nuclei. Immunofluorescence was analyzed using the IX81 microscope  and Xcellence Software (Olympus). For analysis z-stacked images were taken from each sample and foci counted manually. The number of foci in irradiated samples was calculated by background subtraction from non-irradiated controls. All experiments were performed at least two times in duplicates and at least 150 nuclei were counted.For analysis of cell-cycle distribution, cells pretreated with roscovitine (24 h) were fixed at 0, 12, 24 and 48 h after irradiation (6 Gy), stained with propidium iodide and examined by flow cytometry as previously described . The proProteins form whole cell extracts were generated and western blot was performed as previously described . The folIf not stated otherwise, all experiments were performed in triplicates and repeated at least three times. Data are presented as mean values ± standard error of the mean (SEM). Students t-test was used to check for statistical significance with significance level p<0.05. All statistical analysis and graphics were made using Graph-Pad Prism 5.0 program (GraphPad Software).


Small molecule Nutlin-3 reactivates p53 in cancer cells by interacting with the complex between p53 and its repressor Mdm-2 and causing an increase in cancer cell apoptosis. Therefore, Nutlin-3 has potent anticancer properties. Clinical and experimental studies of Nutlin-3 showed that some cancer cells may lose sensitivity to this compound. Here we analyze possible mechanisms for insensitivity of cancer cells to Nutlin-3.genexplain.com) using TRANSFAC® database of transcription factors and their binding sites in genome and using TRANSPATH® database of signal transduction network with associated software such as Match™ and Composite Module Analyst (CMA).We applied upstream analysis approach implemented in geneXplain platform  contains supplementary material, which is available to authorized users. TP53 (gene encoding p53 proteins) (https://www.ncbi.nlm.nih.gov/pubmed/25730903). There is nevertheless a wide range of sensitivity to the Mdm2/p53 binding inhibitors among TP53 wild-type cancer cell lines, which vary widely for different inhibitors (which in turn clearly emphasizes differences of the particular molecular mechanisms of action of different Mdm2-p53 inhibitors) [One of the important targets in cancer cells is well known tumor suppressor protein p53. Among known candidate small molecules interacting with p53 and, therefore, counteracting proliferation of cancer cells is the cis-imidazoline analog compound called Nutlin-3 , 2. Throibitors) .Experimental identification of activated pathways and corresponding potential drug targets in cancer cells is time consuming and very expensive. Computational analysis of gene expression data can help to identify few candidate pathways that can be validated experimentally in focused experiments. Many of such gene expression data are deposited in databases such as ArrayExpress  or Gene But, such approaches provide only a very limited clue to the causes of the observed phenomena and therefore not very useful for selection of potential drug targets. To overcome such limitations we introduced earlier a novel strategy, the “upstream analysis” approach for causal interpretation of the gene expression signatures and identification of potential master regulators –13. ThisMany tumor cells are characterized by a substantial increased expression of p53 inhibitor Mdm2 . In theshttps://clinicaltrials.gov/ct2/show/NCT03041688) and HDM201 . No gene expression data on these two compounds are available in GEO yet. We hope that our current study of Nutlin-3 insensitivity mechanisms will help to prepare the background for future studies of such more potent compounds as AMG-232 and HDM201.Among these compounds, Nutlin-3 is the most commonly used in the anti-cancer studies. Pre-clinical trial data of Nutlin-3 for the treatment of acute myeloid leukemia , 24 has 50 (inhibitory concentration) parameter (concentration of compound leading to 50% of the cell death in the culture). The IC50 parameter was determined using a resazurin viability test. We found that lung cancer cell lines are significantly differing from each other in respect to the sensitivity to Nutlin-3.In order to analyze the effect of Nutlin-3 on lung cancer cells and understand the factors rendering these cells sensitive or insensitive to the treatment we performed a focused study of its biological activity in various lung cancer cell lines carrying wild-type TP53 gene as well as TP53 inactivating mutations. A total of 8 cell lines were analyzed: A549, NCI-H292, A427, COR-L23, DV-90, NCI-H1395, NCI-H1944, NCI-H2228. The cytotoxic activity of Nutlin-3 on lung cancer cell lines was determined based on the IChttp://my-genome-enhancer.com) implemented using BioUML driven systems biology platform . We revealed a number of differentially expressed genes (DEGs) between sensitive and insensitive cell lines. Promoter and pathway analysis of these DEGs using “upstream analysis” approach [Based on our drug sensitivity measurements, we chose one of the most Nutlin-3 sensitive cell lines - H1944, and two less sensitive  cell lines - A427 and NCI-H292 for further studies. We performed microarray experiments on these cell lines before and after treatment by Nutlin-3 in order to reveal different gene expression profiles in sensitive and insensitive cell lines. Comparative analysis of microarray data of these three cell lines was done using a computational pipeline “From genome to target” (approach  helped uhttps://radiopaedia.org/articles/mucoepidermoid-carcinoma-of-lung). The characteristics of these eight cell lines are given in the Table In our study we used the following eight lung cancer cell lines, seven of them are classical non-small cell lung cancer (NSCLC) cell lines  and one is cell line of the mucoepidermal lung carcinoma (NCI-H292), which is also classified as NSCLC according to the existing classification , Cayman Chemical) at concentrations of 34 μM, 17 μM, 8.5 μM, 4.25 μM, 2.2 μM and 0 μM (control).Each concentration point was represented in triplicates. 48 h after the addition of the compound the proportion of viable cells was determined by a viability test with resazurin.Resazurin is an oxidation-reduction dye and is used to analyze the toxicity of compounds in cell culture . The ana50 value was calculated from these curves obtained using the Probit Analysis 1.0 software [The viability test was performed 48 h after the cell culture was incubated with the test compound. To do this, the culture medium with the test compound was removed from the wells of the plate and 500 μl of Dulbecco’s solution (1 ×) containing rezazurin  at a concentration of 50 μg / ml was added. A 500 μl Dulbecco solution with resazurin (50 μg / ml) added to an empty well of a 24-well culture plate was used as control (background). The time of incubation of cells with resazurin was 1 h. The incubation was carried out under sterile conditions, at 99% humidity, at 37 °C and at a concentration of 5%. Accumulation of resorufin (reduced form of resazurin) was assessed by fluorescence detection in the range of 587/607 nm. To assess the results of the test, a calibration curve was constructed for the change in fluorescence as a function of the number of cells. Using this calibration curve we determined the relationship between the intensity of fluorescence and the number of viable cells in culture. The proportion of surviving cells was used to construct the dose-effect curve for each cell line. The ICsoftware .The cells of the lung cancer lines were lysed with Trisol reagent (Invitrogen). The total RNA was isolated according to the recommendations of Invitrogen. The quality of RNA preparations was assessed by electrophoretic separation in a 1.3% agarose gel.The reverse transcription reaction was performed at 42 °C for 45 min in 20 μl of a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 10 mM DTT, 50 mM KCl, 0.2 mM dNTP, Stat-9 primer (10 ng / Μl), 100 unit act., DNA-dependent RNA polymerase MoMLV  and 500 ng of the total RNA.Amplification reactions were performed using thermal cyclers with an optical unit for detecting the fluorescence of iQ5 iCycler or CFX96 (Bio-Rad). The possibility of recording fluorescence in real time was achieved by adding to the reaction mixture the intercalating dye SYBRGreen I.https://www.ncbi.nlm.nih.gov/geo/). In order to detect differentially expressed genes the raw microarray data were normalized and further analyzed using Limma tools from the R/Bioconductor package integrated into the BioUML/geneXplain driven pipeline “From genome to target”. The Limma has calculated LogFC between mean expression values of the genes in two insensitive cell lines in comparison with the sensitive cell line (the logarithm on the basis of 2 of the fold change between different conditions) and the p-value and adjusted p-value (corrected to the multiple testing). In order to take into account the correlated nature of technical replicates (duplicates), which were done for each of the cell lines, we applied the “block” option in the Limma analysis in order to compute the p-values with better precision. . Three cell lines: А427, H292 and H1944 were treated by Nutlin-3 in a concentration that maximally discriminates the sensitive and moderately insensitive cell lines (5 μM) and also in the maximal cytotoxic concentration (30 μM). The cells were incubated with the compound during 24 h. The experiment was done in two biological replicates for each condition. The raw data files with measured gene expression are deposited in the GEO (http://genexplain.com/transfac). The motifs are specified using position weight matrices (PWMs) that assign weights to each nucleotide in each position of the DNA binding motif for a transcription factor or a group of them.Transcription factor binding sites in promoters of differentially expressed genes were analyzed using known DNA-binding motifs described in the TRANSFAC® library , releaseRYN as defined in Eq. (The pipeline “From genome to target” provides tools to identify transcription factor binding sites (TFBS) that are enriched in the promoter regions under study as compared to a background sequence set such as promoters of genes that were not differentially regulated under the condition of the experiment. We denote study and background sets briefly as Yes and No sets. The algorithm for TFBS enrichment analysis, called F-Match, has been initially described in . Brieflyd in Eq.  under thSites and Seq are the sites and sequences counted in Yes and No sets. High Yes/No ratio indicates strong enrichment of binding sites for a given PWM in the Yes sequences. The statistical significance is computed as follows:In Eq. , Sites aP-value < 0.01. The pipeline further maps the matrices to respective transcription factors, and generates visualizations of all results.This binding site enrichment analysis is carried out in the Site Analysis module of the pipeline “From genome to target”. We consider for further analysis only those TFBSs that achieved a Yes/No ratio > 1 and a http://genexplain.com/transpath) [We searched for master regulator molecules in signal transduction pathways upstream of the identified transcription factors using appropriate tools of the pipeline “From genome to target”. The master-regulator search uses the TRANSPATH® database (anspath) . A compranspath) . The goahttp://my-genome-enhancer.com) implemented using BioUML driven systems biology platform . The pipeline is capable of taking various multi-omics data  and automatically performing the “Upstream analysis” [www.genexplain.com/humanpsd) database about known disease relevance of these proteins, are taken into account for such prioritization. Finally, the pipeline “From genome to target” reports a short list of most promising targets.Comparative analysis of microarray data of these three cell lines was done using a computational pipeline “From genome to target” .With the help of the viability test with resazurin we constructed the curves of the percentages of survived cells under increasing concentration of Nutlin-3 and identified IC50 values of Nutlin-3 in the eight lung cancer cell lines . We obtained the following results.50 = 4.9 ± 7.6). The other cell lines showed very different cytotoxic effect of Nutlin-3: H1395 (IC50 = 11.4 ± 3.8), DV-90 (IC50 = 12.9 ± 7.6), COR-L23 (IC50 = 15.1 ± 4.6), H292 (IC50 = 15.4 ± 2.6), A427 (IC50 = 18.8 ± 3.4). The A549 and H2228 cell lines were quite resistant to Nutlin-3, IC50 values were 31.2 ± 5.2 and 33.1 ± 6.7, respectively. Interestingly, that the cell line H2228 which carry the p53 inactivating mutation indeed has demonstrated the highest resistance to Nutlin-3. For the further analysis we selected three cell lines: one, which was the most sensitive to Nutlin-3 – the H1944 cell line, and two, which were still reacting to Nutlin-3, but only under relatively high concentrations – H292 and A427 (moderately insensitive). In the Fig. The H1944 line was the most sensitive to the action of Nutlin-3  of the observed fold change In the Additional file In order to get a comprehensive gene expression profile of the studied cell lines before and after treatment by Nutlin-3 we applied Illumina microarrays (Human HT-12 v3 Expression BeadChips). We treated three cell lines: А427, H292 and H1944 by Nutlin-3 in a concentration which maximally discriminates the sensitive and insensitive cell lines (5 μM) and in the maximally cytotoxic concentration (30 μM)  that gives the end point in the dose-effect curve where no differences in survival between all cell lines were observed,. After data normalization we applied Limma tools (R/Bioconductor package integrated into the pipeline “From genome to target”) and compared gene expression in the insensitive cell lines (A427 and H292) with gene expression in the sensitive cell line (H1944) before treatment and after treatment by two concentration of Nutlin-3. Limma has calculated LogFC (the logarithm on the basis of 2 of the fold change between different conditions), the In order to study the behavior of genes in the insensitive and in the sensitive cell lines after treatment of these cells by 5 μM and 30 μM of Nutlin-3 we computed the LogFC for the change of gene expression in these cell lines before and after the treatment. In Table GO analysis of these differentially expressed genes clearly confirms the well known molecular mechanism of action of Nutlin-3. In the Additional file r = 0.69).In the plot bellow Fig.  we compar = 0.776).We also did the same comparison for the cells treated by 30 μM of Nutlin-3 (data not shown). We detected even higher similarity between gene changes in the sensitive and the insensitive cell lines . Namely the following genes important for this biological process: CDIP1, DPYSL4, DRAM1, TNFRSF10B, TP53INP1 found to be upregulated in the insensitive cell lines and not in the sensitive cell lines. We identified 219 genes that statistically significantly changed their expression (120 up and 99 down) in the insensitive cell lines upon treatment by 30 μM of Nutlin-3 and did not change their expression in the sensitive cell line. , “response to endoplasmic reticulum stress”  and for down-regulated genes - “cell cycle process” , “organic cyclic compound binding”  and “nucleic acid metabolic process” . This analysis shows that several genes that belong to these pathways have got different dynamics of the change of their expression upon treatment by Nutlin-3 in sensitive and in-sensitive cell lines. These differences can be potentially used as promising “dynamic” biomarkers of the sensitivity of the cancer cells to the treatment by Nutlin-3. Still, the causative mechanism of these differences remains rather unclear.Still, in order to reveal genes that differently changed their expression between insensitive and sensitive cell lines we applied filtering of the genes that statistically significantly changed their expression in opposite direction or genes that significantly changed expression in one type of cell lines but were non-changed in the other type. In both comparisons, upon treatment by 5 μM and 30 μM of Nutlin-3 no genes were found that would change their expression in opposite directions. Upon treatment by 5 μM of Nutlin-3 very few genes were found to be differentially expressed, and among them only 26 genes that statistically significantly changed their expression (21 up and 5 down) in insensitive cell lines upon treatment by Nutlin-3 and did not change their expression in the sensitive cell line.  and after treatment by Nutlin-3 (in two concentrations). Such static comparison of the gene expression profiles can help us to detect stably active pro-survival pathways in these cells that protect these cells from cytotoxic activity of Nutlin-3.p-value< 0.05, LogFC> 0.58 for genes with increased expression and LogFC<− 0.58 for the genes with decreased expression).In Table One can see that the number of differently expressed genes between sensitive and insensitive cell lines is rather stable in all three conditions.n = 4, p < 1.7*10− 5), “metabolic process”  and “cell death” . Among the 272 common genes with decreased expression some more specific GO terms showed up, such as: “oxidation-reduction process” , “lipid metabolic process”  and, most interestingly, “positive regulation of cell death” . In the last category, among the genes with most decreased expression in the insensitive cell lines, we can mention such important genes as, S100A9, MLLT11, OSGIN1, CCL5, DAPK1, TNFRSF1A, SMAD3, LYN. Most probably, the low expression of these genes that are able to positively regulate the cell death process promotes the decreased sensitivity of the respective cell lines to the cytotoxic activity of Nutlin-3.Comparison between revealed deferentially expressed genes in these three conditions is shown by Venn diagrams Fig. . One canWe performed gene set enrichment analysis (GSEA) of the obtained three gene expression profiles of differences between sensitive and insensitive lung cancer cell lines. For that we used geneXplain platform and applied the pathways ontology of TRANSPATH® database. The most enriched pathways are presented in the Additional file In the Fig. In the Additional file From the “dynamic” and “static” analysis results we may conclude that although we can detect a number of genes that play their definite role in the “dynamic” creation of cell insensitivity to the drug after the treatment, still the major differences in gene expression of hundreds of genes in these cell lines is observed in “static” comparisons of these cell lines. Moreover, in the “static” analysis the “pre-treatment” comparison of the cell lines delivers the maximum number of enriched pathway. Therefore, in the following analysis we pay our main attention to the genes that we found differentially expressed between insensitive and sensitive cell lines before their treatment by Nutlin-3. We think, that such pre-treatment analysis is most promising in terms of clinical applications, since it can be done on the tumor samples before any treatment and can be used for further definition of the proper anti-cancer therapies.http://genexplain.com/transfac). In this study we used «Genome Enhancer» module of the pipeline «From genome to target» to identify transcription factor binding sites (TFBS) that are enriched in the promoter regions under study as compared to a background sequence set such as promoters of genes that were not differentially regulated under the condition of the experiment. We denote the study and background sets briefly as Yes and No sets. The «Genome Enhancer» module uses two algorithms for TFBS enrichment analysis: F-Match [Transcription factor binding sites in promoters of differentially expressed genes were analyzed using known DNA-binding motifs described by PWMs in the TRANSFAC® library, release 2017.2  ( F-Match  and CMA  F-Match . F-Match F-Match ) and regp-value< 2.53*10− 9). 62 of these genes belong to the biomarkers of “Causal Lung Neoplasms” .  in comparison to the Nutlin-3 sensitive cell line H1944. We focused our attention on the up-regulated genes in order to reveal the potential resistance-specific positive feedback loops (as it is described in our previous paper ) that ar and H292Promoters were extracted from the human genome (build hg38) -1000 nucleotide upstream of TSS (start of transcription) and + 100 downstream. The result of F-Match analysis is presented in the Additional file We also have applied the СМА  algorithm, which allows predicting the formation of complexes of transcription factors binding sites that could jointly regulate groups of genes, such as up-regulated in our study. As a result of application of the CMA algorithm we identified the potential complexes of TFs that may synergistically bind to promoters of these genes and maintain their elevated expression, which in turn leads to the observed relatively high resistance of these cell lines to Nutlun-3.Results of the CMA analysis are shown in the Fig. We identified a combination of PWMs that discriminates the promoters of Yes and No sets rather well. This combination contains PWMs for such important cancer-related transcription factors as p53, E2F-1, EGR1, AP-1, OCT1 and others. The full list of 22 transcription factor genes encoding TFs of selected combination of PWMs is given in the Additional file http://genexplain.com/transpath) [We searched for master regulator molecules in signal transduction pathways upstream of the identified transcription factors. The master-regulator search uses the TRANSPATH® database (anspath)  and is ianspath) . The goaFigure By combining the results of gene set enrichment analysis (see above) and the master regulator search we found especially interesting that identified earlier PI3K sub-pathway (PI3K ---Mdm2-−−/ p53) involves the identified here master-regulator mTOR for inhibition of activity of p53, which is the main target of Nutlin-3 (in the complex with mdm2).Taking all computational evidences together we choose the master-regulator mTOR as the target molecule in the context of PI3K pathway. We applied specific chemical inhibitors in order to test their effect on the survival of the selected cell lines. We choose the following chemical inhibitors Table .Table 5TTo validate the effect of the inhibition of mTOR in the context of PI3K pathways we chose the dual inhibitor I3 that is known to effectively inhibit both PI3K and mTOR action. To test the mTOR-specificity of the effect we used the general inhibitor of PI3K (I1). And as a negative test we used the inhibitor of Bcl-XL (I2) that is known to be involved in cancer but was not predicted here as a master-regulator.I1 in concentrations: 50 μM, 25 μM, 12,5 μM, 6,25 μM, 3,1 μM and 0 μM (Control);I2 in concentrations: 80 μM, 40 μM, 20 μM, 10 μM, 5 μM and 0 μM (Control);I3 in concentrations: 50 μM, 25 μM, 12,5 μM, 6,25 μM, 3,1 μM and 0 μM (Control).We use the following treatment conditions for these three inhibitors.The concentration of the inhibitors was chosen according to their maximal solubility in the medium. DMSO was used to prepare the needed concentration of the inhibitors and the concentration of DMSO was the same in all tested solutions (including Control) and it was below 0.5%. Each measurement was done in three replicas. After 48 h from adding of the inhibitor we measured the percentage of the survived cells using the test with rezuverin.We found that inhibitors I1 and I2 do not demonstrate any cytotoxic activity on all tested cell lines (data not shown). Under any tested concentrations of the inhibitors the number of the survived cells were the same as in the Control and did not differ significantly from 100%.In contrast, the inhibitor I3 demonstrated a moderate cytotoxic activity with the following IC50 values in tested cell lines: A427 (IC50 = 11.8 ± 3.4), H292 (IC50 = 6.05 ± 2.1), DV-90 (IC50 = 19.9 ± 4.6), Н1944 (IC50 = 34.9 ± 3.6) и Н2228 (IC50 = 27.1 ± 6.6). For the three cell lines of our interest the dose-effect curves are shown in Fig. We found that Nutlin-3 insensitive cell lines (A427 and H292) exhibit the highest sensitivity to the dual chemical inhibitor of mTOR-PI3K whereas the Nutlin-3 sensitive cell line (H1944) appeared to be relatively insensitive to this inhibitor. These results confirmed our prediction of the master regulator mTOR in the PI3K signaling pathway, which, most probably, is responsible for the low sensitivity of particular lung cancer cell lines to treatment by the p53-reactivating compound Nutlin-3.50 = 0.49 μM for H292 and 0.55 μM for A427) [p-value < 10− 5). Therefore we found no evidence of the influence of CRTC1-MSML2 fusion on the resistance mechanism to Nutlin-3. It seems that the molecular pathways that are involved in oncogenic program maintained in such cancer subtypes through the CRTC1-MSML2 fusion are rather different from the pathways maintaining the resistance of these cells to the cytotoxic effect of Nutlin-3.Resistance and low sensitivity to chemotherapy and targeted therapy of the cancer cells is one of the biggest problems of cancer treatment. In this work we studied the molecular mechanisms of low sensitivity of cancer cells to the p53-reactivating compound Nutlin-3 using genome-wide transcriptomics profiling followed by causative computational analysis. In order to analyze the effect of Nutlin-3 on lung cancer cells and understand the mechanisms of low sensitivity of some of them to the treatment, we performed an extensive study of the biological activity of the compound Nutlin-3 on a number of lung cancer cell lines. We pay especial attention to the lung cancer cell lines that although caring wild-type TP53 gene  are still very different in their sensitivity to Nutlin-3 treatment. We identified that the cell line H1944 is the most sensitive to the treatment by Nutlin-3. Even under the lowest concentration of the compound the cells of this cell line were rapidly dying. Other cell lines, such as A427 and H292 showed relatively low sensitivity to the treatment by Nutlin-3. The death of cells was triggered only under relatively high concentration of the compound. It is interesting to see that both cell lines A427 as well as NCI-H292 demonstrated rather high sensitivity to another p52-Mdm2 inhibitor AMGMDS3 (ICor A427) . So, it or A427) , which ior A427) . We founIn order to identify such pathways we searched for potential master regulators applying our upstream analysis approach. From the perspective of searching for such master regulators we focus our attention first of all on the genes that have higher expression values in the insensitive cell lines compared to the sensitive (up-regulated genes). Analysis of promoters of these genes helped us to identify several transcription factors with enriched binding sites found in co-localized clusters. We believe that such clusters reflect position and composition of very specific enhanceosome that is formed at the promoters of up-regulated genes and controls their elevated expression in the insensitive cell line. On the next step of our study we analyzed signal transduction network upstream of the revealed transcription factors in order to understand the potential molecular mechanism of activation of these transcription factors. The goal of such analysis was to find few master-regulators in this network that might exert their control on the transcription factors found on the first step.Finally, after performing the search for potential master regulators, we checked which of them were actually up-regulated by themselves. So, we require that genes, which are expressing proteins that were found by the algorithm as potential master regulators, should have significantly higher expression in the insensitive cells compared to the sensitive cells. This reflects the presence of positive feedback loop in the system. We hypothesized that the observed increase of resistance might be supported by the presence of positive feedback loops. We can observe such loops in the network when the genes, expressing master-regulator proteins, are working under control of the transcription factors, which receive activating signals through the signaling cascade, containing proteins, which are expressed by these genes (master regulators). Therefore, the up-regulation of the genes, encoding master regulators in this analysis, indicates the presence of such feedback loops. We think that such positive feedback loops can contribute to the stabilization of the resistance to Nutlin-3 , since As a result, we revealed the following master regulator genes: MTOR, CDK1, CDK2, CHK1, cyclin-B1. We noticed that many of the suggested master regulators are very important proteins that are known to be involved in regulating such processes as cell cycle and apoptosis.p-value < 0.01)  described in the paper Saiki et al.  with theTaking all this into account, we performed experimental validation of the computational predictions. We found that Nutlin-3 insensitive cell lines (A427 and H292), in turn, exhibit the highest sensitivity to the dual chemical inhibitor of mTOR-PI3K, whereas the Nutlin-3 sensitive cell line (H1944) appeared to be relatively insensitive to this inhibitor.The results of this work confirmed our prediction of the master regulators in the mTOR-PI3K signaling pathway responsible for the low sensitivity of these two particular lung cancer cell lines to treatment by the p53-reactivating compound Nutlin-3. As we predicted, the Nutlin-3 insensitive cell lines appeared to be highly sensitive to the inhibitors of mTOR-PI3K pathway. These findings suggest potential preclinical in vitro study to evaluate combining MDM2-TP53 interaction inhibitor(s) with mTOR-PI3K inhibitor(s) across large panel of TP53 WT cell lines using clinically relevant concentrations of clinically relevant inhibitors.Additional file 1: Figure S1.Figure S2. Comparison of genes belonging to different TRASPATH® pathways. Sixteen overlapping genes are the genes encoding proteasome subunits. PISK pathway is most different from other 3 related pathways of cell cycle control. Figure S3. A diagram of top 9 gene promoters (out of 62) with the results of CMA analysis. Exons are shown as blue boxes. The TF binding sites identified by CMA are shown as colored arrows. Gray background shows the position of the site cluster in the promoter. (DOCX 154 kb)R-script for Limma calculation. It includes options for blocks of correlated measurements . Additional file 2: Table S1. Normalized expression values of all genes with detected expression in the studies conditions and mapped to Ensembl. In the tab “Nsen vs Sen” we give the results of Limma analysis of the LogFC between Nutlin-3 insensitive (Nsen) and sensitive cell lines. (XLSX 3291 kb)Additional file 3: Table S2.p-value< 0.05, LogFC> 0.58 (which corresponds to FC > 1.5) for up-regulated and LogFC<− 0.58 for down-regulated genes). Parameter Sum_Logpval sums up logarithms of p-values for one GO term in different conditions of treatment. It allows to sort GO terms according to their total significance in all conditions. (XLSX 334 kb)GO analysis of all 7 sets of genes - Up- and Down- regulated genes upon treatment by Nutlin-3 in two concentrations 5 μM and 30 μM of Nutlin-3  of the obtained three gene expression profiles of differences between sensitive and insensitive lung cancer cell lines. For that we used geneXplain platform and applied the pathways ontology of TRANSPATH® database. (XLSX 147 kb)Additional file 5: Table S4.Results of mapping of upregulated genes on the HumanPSD® disease ontology for all three conditions of Nutlin-3 treatment. The results contain the number of matched genes with the respective disease the calculated p-value and adjusted p-value of such match. (XLSX 78 kb)Additional file 6: Table S5.List of 62 genes up-regulated in Nutlin-3 insensitive cell lines and matching the disease category “Causal Lung Neoplasms”. This list is used for the promoter analysis. (XLSX 61 kb)Additional file 7: Table S6.Result of F-Match analysis of the promoters of 62 genes up-regulated in Nutlin-3 insensitive cell lines. We show the list of transcription factors whose sites were detected as overrepresented in the promoters. We also show the respective PWMs linked to these transcription factors, the Yes/No ration of the site frequency and the LogFC for the expression of the transcription factor gene. In Tab CMA we give results of CMA analysis. 22 TF were revealed. (XLSX 59 kb)Additional file 8: Table S7.Results of correlation analysis of gene expression in 52 cancer cell lines and their sensitivity (IC50) value towards Mdm2 inhibitor AMGMDS3. We found 168 genes positively correlated with IC50 (insensitivity to the Mdm2 inhibitor) and 227 genes negatively correlated . (XLSX 2438 kb)Additional file 9: Table S8.Pathway analysis of the gene expression correlations using GSEA method and TRANSPATH pathway ontology. (XLSX 103 kb)


Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC–MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways. Although there has been a sustained decline in mortality rates over recent decades, breast cancer (BC) continues to be the most prevalent malignancy among women worldwide and is a major cause of female deaths. A number of associated factors, including age, gender, ethnicity, lifestyle  and genetics, such as mutations in the tumor suppressor genes BRCA1 (mutation on chromosome 17) and BRCA2 (mutation on chromosome 13), have been identified as the most common inherited causes of susceptibility to BC. The BRCA1 and BRCA2 genes encode very large proteins that are expressed in a wide variety of different tissues and are implicated in processes such as DNA repair and recombination, checkpoint control of the cell cycle, and transcription. Families with a high incidence of BC may carry mutations in one or both of these genes, or alternatively, members of these families may have similar lifestyle habits and may have been affected by similar environmental factors1346Metabolomics has emerged as a powerful tool for understanding biological processes that occur in humans, and it has mostly been based on the analysis of biofluids, such as blood, saliva, or urine, to discover new cancer biomarkers or to diagnose a diseaseThe use of cell culture metabolomics enables both the discovery of novel biomarkers of pathological conditions and investigation of the related metabolomic pathways . Many of111213in vivo by BC cells. This finding will improve the knowledge about the origin of VOCs and providing comprehensive information as potential BC biomarkers. This strategy can help reveal novel BC biomarkers that might expand the current understanding of this multi-factorial disease. The GC–qMS analyses allow specific identification of VOCs, while multivariate statistical analysis is able to differentiate and discriminate oncologic from normal cells providing proof-of-principle for the detection of different volatile metabolomic patterns in target cells.In this study, a comparative analysis of the volatile metabolomic signature of BC cell lines  and normal human mammary epithelial cells (HMEC), was carried out, in order to identify BC-specific VOCs and to identify a set of biomarkers that could hopefully be correlated with VOCs released VOCs associated with normal breast cells (HMEC) and BC cell lines  were investigated. The cell lines for the present study were chosen based on their different molecular characteristics : namely,We identified twenty-six VOCs belonging to several chemical groups : namely,From these VOCs, 13 were found to be common in all studied breast cells , 5 were present only in normal breast cells (HMEC), and 2 compounds were identified only in the MCF-7 breast cell line.As can be observed in 18202212Similar results were reported by Peled and collaboratorset al. suggested that breast diseases are associated with increases in oxidative stress and a higher activity of P450The pH is one of the parameters that influences the extraction efficiency of VOCs and therefore it is required an optimization step. This was accomplished by the assessment of volatiles from culture media at different pH. We evaluated the effect of pH on the volatile signature obtained from culture media. At pH 2, the MCF-7 cells had the highest total GC peak area with acids  being the most dominant chemical group. Aldehydes  were the most predominant chemical group in the T-47D and MDA-MB-231 cells . At pH 7one-way ANOVA test was applied to analyze the data matrix. From the identified VOCs, a total of 23 (from cultured flask headspace), 52 (from culture media at pH 2), 34 (from the culture media at pH 7) and 43 (from the culture media at pH 10) showed significant differences (p < 0.05)  .Principal component analysis (PCA) was performed for each condition to identify variables to differentiate the VOCs pattern of the HMEC cells from those of the BC cell lines , and from the VOCs patterns obtained from the culture media at different pH values . The staThese results demonstrated that the scores from the cancer cell lines and those from the normal breast cells exhibited separate trends in the plots. Concerning the other tested conditions, the loading scatterplots of the PCA obtained from the analysis of VOCs from cell culture media at pH 2, pH 7 and pH 10, and respective influence of variables, are showed in Under pH 10, the pattern of differentiation between cell lines is similar to obtained for headspace, in which 3 groups  were formed. The differentiation between cell lines obtained under pH 2 and pH 7 may be due to the alterations of molecular components released under more acidic conditions than those normally present in the culture medium (pH 7.3). However, for differentiation and discrimination between normal breast cell lines and oncological breast cell lines based on the VOCs emitted as close to reality as possible in human cell tissues, partial least squares analysis (PLS) and linear discriminant analysis (LDA) were performed only with data from headspace of cell cultures. The statistical data summary of PLS and LDA are described in Similar to obtained in the PCA, HMEC centroid was clearly differentiated from oncologic breast cell lines, and MCF-7 (triple negative type) was distinguished from two luminal A type cell lines. On the other hand, T-47D and MDA-MB-231 remain very close to each other, which PLS values was 0.0588 and 0.0037, respectively. Regarding the influence of variables on PLS values of cell lines, HMEC was highly influenced by 4-methyl-heptane, tetradecane, benzaldehyde and acetophenone, T-47D and MDA-MB-231 were influenced by cyclohexanone, 1,2,4-trimethylbenzene, ethylbenzene and 1,3-dimethylbenzene, and MCF-7 was influenced by the remaining VOCs.The linear discriminant analysis (LDA) was applied as a supervised pattern recognition method in order to discriminate statistically the cell lines under study, where samples were grouped according to molecular type as follows: N (HMEC), BL (T-47D and MDA-MB-231) and BTN (MCF-7). The LDA scatterplot of cell lines classification according to canonical functions were showed in et al.et al.et al.The cell lines samples formed three clearly defined groups with a classification rate of 100%. Recognition ability, calculated as the percentage of members of the data set that were correctly classified, and prediction ability, calculated as the percentage of members that were correctly classified, were 100% in all cases. After applying LDA with backward removal (p < 0.05) of variables, only two VOCs proved to be significant for discrimination between three defined previously, namely 1,2,4-trimethylbenzene and benzaldehyde. These compounds have been already identified in cancer cell studies by Brunner in vitro VOCs associated with BC to identify potential volatile biomarkers to be used in early diagnosis. The headspace of culture media of normal and cancer cell lines was analyzed at different pH conditions. Sixty VOCs were identified as belonging to several chemical groups: namely, alkanes, aldehydes, ketones, acids, alcohols and benzene derivatives. Most of the identified VOCs are common to all BC cell lines and normal human mammary epithelial cells, but six of them, 2-pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate, and 2-methyl butanoate, were detected only in the headspace of cancer cell lines. Multivariate statistical data obtained in this study revealed that combining in vitro assays with HS-SPME/GC-MS is a useful strategy to differentiate and discriminate the volatile metabolomic signature of normal cells and BC cell lines according to molecular type, thus contributing to the discovery of novel biomarkers of BC and investigations of the related metabolomic pathways, and thereby improving the diagnostic tools for BC.This study demonstrated that HS-SPME/GC-MS is a simple, rapid, sensitive and solvent-free method that can be used to establish the volatile metabolomic patterns of normal and cancer breast cells. In addition, this study showed the potential of screening the Phosphate buffer saline (PBS) was purchased from Sigma-Aldrich , sodium chloride was obtained from Panreac , the SPME holder for manual sampling of SPME fiber [50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)] and the glass vials were purchased from Supelco . The SPME fiber was conditioned according to manufacturer’s instructions. Before each daily analysis, the fiber was conditioned for 10 min in the injector port to prevent carryover. T75 glass flasks were purchased from Ningbo .®) supplemented with 15% fetal bovine serum , 1% Antibiotic-Antimycotic solution , 1% MEM Non-Essential Amino Acids solution , 1 mM sodium pyruvate  and 10 μg/mL human insulin ; T-47D cell line was grown in 85% RPMI 1640 supplemented with 15% fetal bovine serum (FBS), 1% Antibiotic-Antimycotic solution and 10 μg/mL human insulin, while the MDA-MB-231 cell line was grown in 85% RPMI 1640 supplemented with 15% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic solution. Human mammary epithelial cells (HMEC) were purchased from Life technologies (Gibco®) and grown in HUMEC serum-free medium supplemented with 20 μg/mL of Antibiotic-Antimycotic solution . All cells were incubated in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C. Culture media was changed every 2 days and the cultures were passaged with 0.25% trypsin-EDTA  when 80% of confluence was achieved.The human breast adenocarcinoma cell line MCF-7 and human breast carcinoma cell lines T-47D and MDA-MB-231 were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures . MCF-7 was grown in 90% RPMI 1640  for 30 min and then washed with PBS (3 times). The cells were then cultured in the T75 flasks for 48 h. After this period, volatile metabolites were extracted using a 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fiber exposed in the headspace of the flasks for 45 min at 37 °C, followed by injection into the GC injection port for 10 min to allow the desorption of VOCs from the fiber. After these extractions, cell-free aliquots were collected from the flasks holding 10 mL of the culture medium with growing cells. They were centrifuged to remove any suspended cells, and then 1 mL aliquots were adjusted to pH 2, 7 or 10 with 1 M HCl or 1 M NaOH−1 (hold for 10 min); (c) 150 °C for 10 min; (d) increase temperature until 220 °C, at a rate of 7 °C min−1; and (e) 220 °C for 10 min. Column flow was constant at 1.0 mL/min using helium  as the carrier gas. The injection port was maintained at 250 °C and operated in the splitless mode. Regarding MS analyses, the operating temperatures of the transfer line, quadrupole and ionization source were 270, 150 and 230 °C, respectively. The electron impact mass spectra were recorded at 70 eV and the ionization current was 10 μA, and data acquisition was performed in scan mode (30–200 m/z). The identification of metabolites was performed comparing mass spectra with the Agilent MS ChemStation Software  equipped with the NIST05 mass spectral library with a similarity threshold higher than 80%, or with commercially standards when available. All experiments were performed in triplicate and the results were expressed as the mean ± standard deviation.VOCs in the headspace were analyzed using an Agilent Technologies 6890 N Network gas chromatograph system  equipped with a BP-20 fused silica column  interfaced with an Agilent 5975 quadrupole inert mass selective detector. The following oven temperature profile was set: (a) 5 min at 45 °C; (b) increase temperature until 150 °C, at a rate of 2 °C minone-way ANOVA, and p < 0.05 was considered as statistically significant. PCA, PLS and LDA were carried out on VOCs selected by ANOVA to evaluate differences among the studied groups. PCA was performed in order to obtain differentiation between samples under study without classification and PLS was used as a supervised linear pattern recognition algorithm for data classification of samples. PCA and PLS were performed through variables values scale by unit standard deviation with convergence criterion (0.0001) and leave-one-out cross validation for accuracy confirmation. For LDA analysis a backward selection method was used with a p < 0.05 through Wilks test. For cross validation a leave-one-out strategy was used.Statistical tests were performed using the StatSoft STATISTICA 12.0 (2013) software . Differences in VOCs between groups were tested with How to cite this article: Silva, C. L. et al. Volatile metabolomic signature of human breast cancer cell lines. Sci. Rep.7, 43969; doi: 10.1038/srep43969 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.