WAF1 and MDM2, the addition of rucaparib did not enhance the p53 activation seen with the MDM2 inhibitors alone. These results demonstrate that the synergistic effect on growth inhibition observed in the combination between rucaparib and Nutlin-3/RG7388 is not the result of increased p53 molecular pathway activation. Nevertheless, combined treatment of Nutlin-3/RG7388 with rucaparib increased cell cycle arrest and apoptosis, which was marked for A2780 and IGROV-1. These data indicate that combination treatment with MDM2 inhibitors and rucaparib has synergistic and dose reduction potential for the treatment of ovarian cancer, dependent on cell type.Ovarian cancer is the seventh most common cancer worldwide for females and the most lethal of all gynecological malignancies. The treatment of ovarian cancer remains a challenge in spite of advances in debulking surgery and changes in both chemotherapy schedules and routes of administration. Cancer treatment has recently been improving with the introduction of targeted therapies to achieve greater specificity and less cytotoxicity. Both PARP inhibitors and MDM2-p53 binding antagonists are targeted therapeutic agents entered into clinical trials. This preclinical study evaluated the effect of Nutlin-3/RG7388 and rucaparib as single agents and in combination together in a panel of ovarian cancer cell lines. Median-drug-effect analysis showed Nutlin-3/RG7388 combination with rucaparib was additive to, or synergistic in a cell type-dependent manner. Mechanism studies showed rucaparib alone had no effect on p53 stabilization or activity. Although treatment with Nutlin-3 or RG7388 induced stabilization of p53 and upregulation of p21 Ovarian cancer is the fifth leading cause of cancer-related female deaths and was reported to be responsible for approximately 152,000 deaths worldwide in 2012 , 4. DiffBRCA1/2 mutation or other HRR defective status cannot efficiently repair these double-strand breaks, leading to cell death [BRCA1/2 or other HRR deficiencies, more than in normal DNA repair proficient cells [BRCA mutation (germline and/or somatic) associated advanced ovarian cancer who have been treated with two or more chemotherapies [The inhibition of PARP enzymatic activity hinders DNA repair via the base excision repair (BER) pathway. For cells deficient in homologous DNA recombination repair (HRR) this results in multiple lethal DNA double-strand breaks normally repaired by the HRR pathway. The tumor cells with ll death –8. Anothnt cells , 10. Rucherapies .50 value of 6 nM [Reactivation of wild-type p53 by preventing the protein-protein binding interaction between p53 and its negative regulator MDM2 induces the growth inhibitory and/or pro-apoptotic functions of p53, and has been demonstrated to have potential as a therapeutic strategy for non-genotoxic activation of p53. Nutlin-3 provided the mechanistic proof-of-concept for small molecule inhibition of the MDM2-p53 interaction and continues to be a useful reference tool compound; however, its potency and pharmacological properties are suboptimal for clinical use , 13. RG7 of 6 nM . These c of 6 nM , 16.TP53 may benefit from MDM2 inhibitor treatment [Up to 50% to 60% of epithelial ovarian cancer is estimated to be deficient in HRR and hence likely to respond to PARP inhibitors . The appreatment . Combinareatment .TP53 ovarian cancer cell lines.In the current study it was hypothesized that combination treatment of Nutlin-3/RG7388 with rucaparib further activates the p53 pathway by inhibition of PARP and results in enhanced induction and stabilization of p53 via Nutlin-3/RG7388 treatment to increase growth arrest and/or apoptosis in wild-type TP53 ovarian cancer cell lines derived from tumors of different histological subtypes [50 values, required concentration of each compound leading to 50% growth inhibition, showed that wild-type TP53 ovarian cancer cell lines were significantly more sensitive to Nutlin-3/RG7388 compared to mutant, which is consistent with their mechanism of action (p<0.0001 Mann-Whitney test)  nM) and micromolar range (1.76 ± 0.51 (SEM) μM) respectively. In contrast, TP53 mutant cell lines had GI50 values greater than 10 μM (17.8 ± 2.9 (SEM) μM) for RG7388 and range 21.2->30 μM for Nutlin-3 (Table A sulforhodamine-B (SRB) assay was used to investigate growth inhibition by Nutlin-3/RG7388 or rucaparib for a panel of wild-type and mutant subtypes –22  Figure . The GI550 values. The GI50 values significantly varied showing a range of responses, with A2780 (3.26±0.47 μM) and SKOV-3 (> 25 μM) as the most sensitive and resistant cell lines respectively. Although CP70 cells are derived from A2780, they have genetic alterations in addition to the difference in TP53 genomic status. In particular, CP70 cells are mismatch repair (MMR)-deficient due to loss of the MLH1 gene and they are resistant to rucaparib even though A2780 cells are MMR-competent and sensitive to rucaparib. The CP70+ cell line is MLH1-corrected by chromosome 3 transfer into the CP70 cells and retains the heterozygous TP53 mutation. The A2780 and derived cell lines differ in growth rate, TP53 status and MLH1 status and show differences in response to cisplatin and rucaparib, they are nevertheless all derived from the same tumor and thus overall are genetically closely related. The results of this study showed no relationship between the status of TP53 and response to rucaparib   for 72 hours to construct growth inhibition curves and calculate the GI) Figure .TP53 ovarian cancer cell lines using median-effect analysis. The sensitivity of the wild-type TP53 cell lines to growth inhibition during 72 hours exposure to rucaparib and Nutlin-3/RG7388 was determined as single agents, and in combination at 5 equipotent concentrations between 0.25× and 4× their respective GI50 concentrations for the A2780 cell line. Owing to the high GI50 for IGROV-1 and OAW42 in response to rucaparib, 3 equipotent concentrations between 0.25× and 1x their respective GI50 concentrations were used to evaluate the combination effect of rucaparib with Nutlin-3/RG7388. The effect of combined treatment was cell type dependent. The combination of Nutlin-3/RG7388 with rucaparib at all concentrations led to greater growth inhibition compared to either agent alone for the A2780, IGROV-1 and OAW42 cell lines, although the increase was smaller for the OAW42 cells  values calculated. CI values for each constant ratio combination and at effect levels of EDd Figure . Combinee Figure .TP53 cell lines were treated with rucaparib, Nutlin-3 or RG7388 alone, and in combination at constant 1:1 ratios of 1/2x and 1x their respective GI50 concentrations for 4 hours. They were also treated with rucaparib, Nutlin-3 or RG7388 alone, and in combination at constant 1:1 ratios of 1x their respective GI50 concentrations for 24 hours, to test the induction of PUMA (BBC3) as a TP53-related pro-apoptotic gene. Western blot analysis showed that rucaparib treatment as a single agent had no effect on p53 stabilization, upregulation of p21WAF1, MDM2 or PUMA compared to DMSO control, nor generally did rucaparib increase the effect of the MDM2 inhibitors on the p53 pathway  and other cell lines  were resistant (GI50 > 10 μM). No relationship was found between the TP53 status of cells and response to rucaparib. These results are in line with those of previous studies reporting the sensitivity of A2780 and resistance of OAW42, IGROV-1, CP70 and SKOV-3 in response to rucaparib [50 values for rucaparib across all cell lines were much higher than the concentration needed to reduce the cell-free enzymatic activity by half, Ki=1.4 nM [in vivo and used in clinical trials [Within the panel of ovarian cancer cell lines studied, wild-type f action . Followif action . Among tucaparib , 30. Ovei=1.4 nM . Howeverl trials , 26, 32.PTEN  (89682879_89682887delAGGGACGAA)  . MDAH-27and KRAS .Overall, the reasons for the greater sensitivity of A2780 and MDAH-2774 to rucaparib are not clear. Further research is needed to identify reliable biomarkers to stratify patients who will benefit from treatment with PARP inhibitors.de novo inactivating TP53 mutations or selection of pre-existing subclones of TP53 mutant cells that might be present as a result of cancer cell genomic instability and tumor heterogeneity [BRCA gene has been confirmed to occur in patients [Resistance to MDM2-p53 binding antagonists has been suggested to be acquired by prolonged exposure of cells to sub-lethal doses through ogeneity , 39. Devpatients . Combinapatients , 41.TP53 status as combined targeted therapeutics. Updated results from the ARIEL2 clinical trial of rucaparib in 152 patients with wild-type BRCA1/2 who were sensitive to platinum reported a response rate of 36% in those with BRCA-like DNA repair deficiency status. Less common mutations involved in Homologous Recombination Deficiency (HRD) and predictive of response to platinum may be present in almost one third (33%) of ovarian cancer patients [TP53 wild-type. Thus nearly 10% of ovarian cancer patients (one third of the 30% not high grade serous) who are likely to be sensitive to both MDM2 inhibitors and rucaparib may benefit from combination treatment with these agents. In addition, almost 4% of high grade serous ovarian cancers nevertheless have wild-type TP53 and these mostly have BRCA1/2 mutation or other HRD status [This study set out with the aim of assessing the effect of combination treatment of Nutlin-3 or RG7388 with rucaparib in a panel of ovarian cancer cell lines of known patients . Almost D status , renderiOverall, the effect of combined treatment was cell type and exposure time dependent, with a higher synergistic effect for the combination of Nutlin-3/RG7388 with rucaparib for A2780 and IGROV-1 cell lines. Interestingly, although both IGROV-1 and OAW42 were resistant to rucaparib, there was nevertheless a synergistic effect for the combination of Nutlin-3/RG7388 with rucaparib. A possible explanation for this is that the defects conferring sensitivity to PARP inhibitors as single agents may be different from those which play an important role in the response to the combination treatment. For example, serious deficiencies in HRR may affect the sensitivity to PARP inhibitors as single agents, while mild defects in HRR may have no effect on response to PARP inhibitors alone but may nevertheless influence the effect of combination treatments . AnotherAn important and clinically relevant finding from the data is the favorable DRI values in both combination treatment of Nutlin-3 or RG7388 with rucaparib. Additive and even mildly antagonistic results of combined treatment can nevertheless be of potential clinical use due to favorable DRI values , 48. TheWAF1, MDM2 and PUMA across all three cell lines. Combination treatment of Nutlin-3 or RG7388 with rucaparib induced stabilization of p53 and upregulation of p21WAF1, MDM2 and PUMA compared to rucaparib on its own, whereas rucaparib had no enhancement of the p53 activation by MDM2 inhibitors alone. These results demonstrate that the synergistic effect on growth inhibition observed for the combination of rucaparib and Nutlin-3/RG7388 is not the result of an accentuated p53 response and hence not related to the p53 molecular pathway. The interplay between PARP and p53 is controversial which may be related to the type of DNA damage, type of PARP inhibitors and intensity of replicative stress [Rucaparib on its own had no effect on p53 stabilization and upregulation of its downstream targets p21e stress , 50.TP53 ovarian cancer cell lines in a time and dose-dependent manner. Rucaparib had little effect on the cell cycle distribution for IGROV-1 and OAW42 cell lines, which is in agreement with the results obtained by Porcelli et al. [Individually, Nutlin-3 and RG7388 induced cell cycle arrest in wild-type i et al.  that indi et al. . Furtheri et al. .WAF1 expression in response to MDM2 inhibitors and its critical role in cell cycle arrest in G2/M by inhibiting mitotic Cdk complexes and Rb phosphorylation [Across all three cell lines, combined treatment of Nutlin-3/RG7388 with rucaparib increased the proportion of cells in the G2/M phase of the cell cycle, which was marked for A2780 and IGROV-1. This can be explained by induction of p21rylation . Also, prylation . The incrylation . CombinaIn conclusion, the present study demonstrates that combination treatment with MDM2 inhibitors and rucaparib has synergistic and/or dose reduction potential dependent on cell genotype and compound and merits further investigation. Monitoring HRR status, cell cycle markers, PARP expression and PARP activity are likely to provide additional useful information to assess the effectiveness of PARP inhibitors. This information may be helpful to stratify the patients who might benefit from PARP inhibitors and their potential combination with the emerging class of MDM2 inhibitors.Nutlin-3, a 1:1 mixture of the active enantiomer Nutlin-3a and the inactive enantiomer Nutlin-3b, was purchased from NewChem  and RG7388 (Idasanutlin) was synthesized by Ruth Bawn and Amy Heptinstall within the Newcastle Drug Discovery Group. Both were dissolved in dimethyl sulfoxide (DMSO). Rucaparib was kindly supplied by Clovis Oncology, and prepared in 10mM stocks solubilized in DMSO.TP53 status and histological subtype are listed in Table TP53 mutation  [MLH1-corrected CP70+ cell line was grown in RPMI-1640 supplemented with 10% (v/v) FBS and Hygromycin B  [TP53 mutation . SKOV-3 and MDAH-2774 cell lines were cultured in DMEM supplemented with 10% and 5% (v/v) FBS and penicillin/streptomycin respectively. As information on the TP53 status of SKOV-3 and IGROV-1 in the literature was contradictory, sequencing was performed. The results of PCR-based Sanger sequencing of TP53 exon 5 and exon 4 confirmed the wild-type TP53 status of the IGROV-1 cell line (TP53 exon 4 and no substitution mutation (c.179 A->G) was detected in TP53 exon 5  [The ovarian cancer cell lines used in this study, their l172Phe) . The MLHs, Inc.) . This ceell line . For the3 exon 5 . The MDAg273His) .50 values, the required concentrations of each compound leading to 50% growth inhibition, were determined by Sulforhodamine B (SRB) growth inhibition assays for drug exposure over 72 hours and the absorbance of the re-dissolved SRB protein stain was measured at 570 nm using a 96-well plate spectrophotometer (Spectramax 250 Molecular Devices) [TP53 cell lines were treated for 72 hours with each agent alone and in combination simultaneously at constant 1:1 ratios of 0.25x, 0.5x, 1x, 2x, and 4x their respective GI50 concentrations. Median-effect analysis was used to calculate Combination Index (CI) and Dose Reduction Index (DRI) values [The GIDevices) . Growth WAF1 1:100 , PUMA 1:1000  and p53 1:500  were used. Secondary goat anti-mouse HRP-conjugated antibodies  were used at 1:1000. All antibodies were diluted in 5% milk/1XTBS-Tween (w/v). Enhanced chemiluminescence (GE Life Sciences) and X-ray film (Fujifilm) were used to visualize the proteins.Lysis buffer  was used to harvest whole-cell lysates, followed by sonication. The concentration of protein in the cell lysates was estimated by using a bicinchoninic acid (BCA) assay. Novex® 4-20% Tris-Glycine 12-well polyacrylamide gradient gels  were used to separate proteins. The separated proteins were transferred by perpendicular electrophoresis to a nitrocellulose Hybond™ C membrane . Monoclonal Mouse Anti-Human primary antibodies Actin 1:1000 , MDM2 1:300 , p2150 concentrations for 24, 48 and 72 hours. Harvested cells, both floating and adherent, were washed with PBS and resuspended in 500 μL PBS with 1mg/mL sodium citrate , 100 μg/mL propidium iodide (Sigma), 200 μg/mL RNAse A (Sigma) and 0.3% Triton-X (Sigma). Samples were analyzed on a FACSCalibur™ flow cytometer using CellQuest Pro software . Cell cycle distribution was determined using Cyflogic .Cells were treated with Nutlin-3, RG7388 and rucaparib alone and with combinations of Nutlin-3 or RG7388 with rucaparib simultaneously at constant 1:1 ratios of 0.5x and 1x their respective GICaspase 3/7 activity was measured using a Caspase- Glo 3/7 assay following the manufacturer’s instructions .All statistical tests presented were carried out using GraphPad Prism version 5.04 software. A p-value of <0.05 was considered to be statistically significant based on at least n=3 experimental repeats.


Proteasome inhibitors have distinct properties and the biochemical consequences of suppressing ubiquitin E1 enzymes and the proteasome differ. We compared the effects of the proteasome inhibitors bortezomib, ixazomib and carfilzomib and the ubiquitin E1 enzyme inhibitor MLN7243/TAK-243 on cell viability and cell death in normal keratinocytes and cutaneous squamous cell carcinoma (cSCC) cell lines. The effects of both a pulse of treatment and more extended incubation were investigated. This is relevant to directly-delivered therapy  where the time of exposure can be controlled and a short exposure may better reflect systemically-delivered inhibitor pharmacokinetics. These agents can selectively kill cSCC cells but there are variations in the pattern of cSCC cell line sensitivity/resistance. Variations in the responses to proteasome inhibitors are associated with differences in the specificity of the inhibitors for the three proteolytic activities of the proteasome. There is greater selectivity for killing cSCC cells compared to normal keratinocytes with a pulse of proteasome inhibitor treatment than with a more extended exposure. We provide evidence that c-MYC-dependent NOXA upregulation confers susceptibility to a short incubation with proteasome inhibitors by priming cSCC cells for rapid BAK-dependent death. We observed that bortezomib-resistant cSCC cells can be sensitive to MLN7243-induced death. Low expression of the ubiquitin E1 UBA1/UBE1 participates in conferring susceptibility to MLN7243 by increasing sensitivity to MLN7243-mediated attenuation of ubiquitination. This study supports further investigation of the potential of proteasome and ubiquitin E1 inhibition for cSCC therapy. Direct delivery of inhibitors could facilitate adequate exposure of skin cancers. Cutaneous squamous cell carcinoma (cSCC) causes significant mortality and morbidity in the general population . In addiUbiquitination regulates protein stability, activity, association and localization , 7. ThisThe proteasome inhibitor bortezomib has been widely used to treat patients with multiple myeloma. Two additional proteasome inhibitors: ixazomib and carfilzomib have recently been approved for multiple myeloma therapy. These agents differ in their affinity for inhibition of the individual proteolytic activities of the proteasome and/or in their reversibility. The importance of these differences in influencing the therapeutic efficacy of these inhibitors is not fully understood. At low concentrations all of these compounds inhibit the chymotrypsin-like activity of the proteasome. Bortezomib and ixazomib inhibit the caspase-like activity at moderately high concentrations and the trypsin-like activity at high concentrations . ModeratAn alternative way of targeting the core ubiquitin-proteasome system is to inhibit the ubiquitin E1 enzymes . BlockinThe therapeutic potential for cSCC of targeting the ubiquitin-proteasome system has not been adequately explored. Furthermore, the anti-tumour activity and the degree of tumour selectivity of proteasome and ubiquitin E1 inhibition have not been compared for any cancer type. It is common in pre-clinical investigations to determine the effect of extended exposure to proteasome inhibitors on the viability of cultured cells. It is relevant for directly-delivered therapy to assess the effects of different times of tumour treatment. It may also better model systemically-delivered inhibitor pharmacokinetics to investigate the consequences of a shorter pulse of inhibitor treatment -24, 39. We investigated whether there were differences in responses to the proteasome inhibitors bortezomib, ixazomib and carfilzomib and the ubiquitin E1 inhibitor MLN7243. The effects of these agents were assessed in untransformed normal keratinocytes from an RDEB patient (RDEBK), normal human keratinocytes (NHK) and eight cSCC-derived cell lines. cSCCs arise from the malignant transformation of keratinocytes in the epidermis. Keratinocytes constitute over 90% of cells within this layer of the skin. It is consequently appropriate to include keratinocytes in the study as the cell of cSCC origin and a major normal cell type that would be exposed to locally-delivered therapy. Measurement of the proteolytic activities of the proteasome verified target suppression by proteasome inhibitors Figure . In addiClonogenic assays indicated that a 6 to 8-hour treatment with the inhibitors was required for efficient anti-cSCC activity . To compWith continuous exposure to bortezomib there was little selectivity for cSCC compared to untransformed keratinocytes Figure . HoweverThe general pattern of ixazomib susceptibility in normal keratinocytes and representative cSCC cell lines was similar to that of bortezomib . Under tCarfilzomib was more generally selective for killing cSCC cells compared to normal keratinocytes than the other agents tested Figure . The cSCThe ubiquitin E1 inhibitor MLN7243 selectively killed a subset of cSCC lines . In bortezomib-sensitive SCCRDEB4 and SCCIC1 cells siRNA-mediated depletion of c-MYC and the pro-apoptotic proteins NOXA and BAK was found to attenuate cell death in response to a pulse of bortezomib Figure . BAK is Basal expression of c-MYC and NOXA was low in bortezomib-resistant normal keratinocytes and SCCIC1Met cells and BAK levels were low in the bortezomib-resistant SCCRDEBMet cell line Figure . Low expc-MYC/NOXA/BAK also contribute to promoting rapid cell death in response to a pulse of carfilzomib treatment in bortezomib-sensitive cSCC cells . In carfTo identify determinants of sensitivity to MLN7243 the protein expression of the ubiquitin E1 UBA1 was compared in the panel of cells used in this study Figure . Two majKnockdown of UBA1 did not greatly alter the pattern of bulk high molecular weight ubiquitin conjugates in cSCC cells, even in the SCCRDEBMet line which expresses the lowest endogenous levels of the E1 Figure . In addiWe compared the effects in normal keratinocytes and cSCC cell lines of a long and short treatment with proteasome inhibitors and the ubiquitin E1 inhibitor MLN7243. We show that a pulse of proteasome inhibitor exposure results in greater selectivity for killing cSCC cells than a more extended exposure. In sensitive cSCC cell lines a pulse of bortezomib treatment gives the highest level of selectivity compared to normal keratinocytes but some cSCC cell lines are bortezomib-resistant. These bortezomib-resistant tumour cells can be sensitive to MLN7243. Carfilzomib is more generally cSCC selective than bortezomib or MLN7243. We provide evidence that c-MYC, NOXA and BAK are determinants of susceptibility to a pulse of proteasome inhibition and that low UBA1 expression confers susceptibility to MLN7243.This study indicates that the cSCC selectivity of a pulse of proteasome inhibitor exposure is related to a differential dependency for survival of sensitive cSCC cells and normal keratinocytes on the proteolytic activities of the proteasome. For example, killing of the most bortezomib-sensitive cSCC cell lines by a short treatment with bortezomib occurs at inhibitor concentrations that markedly attenuate the chymotrypsin and caspase-like activities but only modestly inhibit the trypsin-like activity. In normal keratinocytes death is associated with inhibition of all three activities at high bortezomib concentrations. This suggests that the window of selectivity for cSCC cells compared to normal keratinocytes can be determined by the specificity of inhibitors for the activities of the proteasome. For instance, a small molecule which inhibits the chymotrypsin and caspase-like activities without substantially inhibiting the trypsin-like activity may show even greater selectivity for sensitive cSCC cells than bortezomib. These results are consistent with the previous finding that inhibition of different individual proteolytic activities and combinations of proteolytic activities of the proteasome results in distinct degrees of interference with protein degradation , 16. TheWe observed that concentrations of bortezomib and carfilzomib that kill cSCC cell lines have divergent effects on the proteolytic activities of the proteasome. Variations in the extent to which cellular pathways are affected due to these differences in proteasome inhibition may underlie the greater cSCC selectivity of continuous exposure to carfilzomib and the ability of a pulse of carfilzomib to kill cSCC cells resistant to a short treatment with bortezomib. Consistent with this, our study indicates that a pulse of carfilzomib can induce cell death by different mechanisms in bortezomib-resistant and bortezomib-sensitive cells. Differences in the mechanisms through which distinct proteasome inhibitors kill cells have been observed previously .NOXA transcription. Upregulation of basal NOXA mRNA expression by elevated c-MYC could contribute to fast protein accumulation upon inhibition of the proteasomal degradation of NOXA. Proteasome inhibition also increases c-MYC transcriptional activity towards the NOXA gene [c-MYC, NOXA and BAK promote cell death in response to an extended exposure to bortezomib in a number of cancer types , 62, 63.OXA gene , 57, 68.The pattern of sensitivity of cSCC cells to ubiquitin E1 inhibition is different from proteasome inhibitors. The two cSCC cell lines most resistant to a pulse of bortezomib are among the most sensitive to MLN7243-induced cell death. We observed that low expression of both UBA1A and B isoforms is associated with MLN7243 sensitivity and high expression of UBA1A with MLN7243 resistance. Furthermore, knockdown of UBA1A and B confers MLN7243 sensitivity to resistant cSCC cells. MLN7243-induced cell death is associated with a reduction in the level of ubiquitin conjugates. A lower concentration of MLN7243 is required to diminish ubiquitination in cSCC cells with low UBA1 expression. UBA1 protein levels may thus provide a marker for tumour sensitivity to MLN7243. It would be of great interest to determine the mechanisms responsible for the observed differences in UBA1A and B expression. A better understanding of how levels of UBA1 isoforms are regulated could lead to the development of therapeutic interventions that modulate their expression. This may provide a means to enhance tumour sensitivity or increase the resistance of normal cells to MLN7243. UBA1 and UBA6 are both inhibited by MLN7243. While our study supports a pre-eminent role of UBA1 in determining the sensitivity of cSCC cells to MLN7243 under some circumstances suppression of UBA6 could contribute to the anti-tumour activity of this inhibitor. UBA6 can play a non-redundant role in maintaining cell viability , 69.A high degree of E1 suppression is required to reduce bulk high molecular weight ubiquitin conjugates in the cSCC cell lines examined. This indicates that the ubiquitin E1s are not normally rate-limiting for these ubiquitination events. This is consistent with previous studies . UBA1 isThis study indicates that there is therapeutic potential for proteasome and UBA1 inhibition for cSCC. Future work could initially be aimed at developing direct delivery of inhibitors to tumours. This would overcome limitations of systemic delivery and allow optimal exposure of cSCCs. Effective directly-delivered therapy would be of benefit to cSCC patients and it would inform systemic therapy for treating cSCC and other solid tumours.2 in a humidified atmosphere in keratinocyte medium containing 10% serum and growth factors [Normal keratinocytes (NHK and RDEBK) and cSCC cell lines were isolated and maintained as described , 72. Cel factors . cSCC ce factors . SCCIC1/Inhibitors were dissolved in DMSO (20 to 50 mM stocks). Unless otherwise indicated cells were treated with inhibitors 16 to 24 hours after plating. Compounds used in this study were: bortezomib , carfilzomib , ixazomib (S2180: Selleckchem) and MLN7243 .Dharmacon ON-TARGETplus modified siRNAs  were used in this work and are listed in Cells were seeded into 96 well plates and live cell number and cell death were analysed according to the manufacturers’ instructions with an IncuCyte ZOOM real-time imager  using the CellTox Green Cytotoxicity Assay . Relative EC50 values were determined using GraphPad Prism . For clonogenic assays cells were seeded into six well plates. Inhibitors were added for the indicated times and then cells were maintained in drug-free medium for up to 2 weeks to allow colony formation. Colonies were fixed in 10% methanol, 10% acetic acid and stained with crystal violet.The statistical significance of differences between two groups (control and targeting siRNA) was determined using a one tailed T-test.The three proteolytic activities of the proteasome were measured in parallel in cells seeded in 96 well plates using the Cell-Based Proteasome-Glo Assay (G1180: Promega). Medium from wells containing no cells was used as a blank.The primary antibodies used are listed in


Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production  were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities.Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of  Polygonum multiflorum Thunb. is a member of the family Polygonaceae and a traditional Chinese medicinal herb. It has been used in the preparation of herbal medicines in China, Japan, Korea, and Taiwan, as it has important pharmacological functions  × 100.Two human cancer cell lines, namely, colon HT-29  and estrogen-dependent breast MCF-7 (human breast adenocarcinoma) cancer cell lines were purchased from the KCLB  and were used for cytotoxicity screening of the lyophilized cell suspension powder extracts  in p ≤ 0.05). All statistical tests were performed using the SPSS Ver. 20  statistical software package.Each experiment was repeated three times. The data were expressed as mean ± standard deviation (SD) values. One-way analysis of variance and Duncan’s multiple range test were used to determine significant differences  was combined with elicitors, 100 µM JA or SA. The levels of phenolic groups such as flavonols and hydroxybenzoic and hydroxycinnamic acids were higher in the JA- and SA-elicited culture cells than in the control cell cultures. The total phenolic and flavonoid contents and antioxidant, antimicrobial, and anticancer activities were higher in the JA- and SA-elicited cell cultures than in the control cell cultures. Our protocol will be useful for biochemical and bioprocess engineering and sustainable production of valuable phytochemicals (AQs and phenolic compounds) via cell suspension culture.For the first time, we successfully established cell suspension cultures of


Arabidopsis. However, our knowledge of how formation of the root system is altered in response to diverse inductive cues is limited. Here, we show that WOX11 contributes to root system plasticity. When seedlings are grown vertically on medium, WOX11 is not expressed in LR founder cells. During AR initiation, WOX11 is expressed in AR founder cells and activates LBD16. LBD16 also functions in LR formation and is activated in that context by ARF7/19 and not by WOX11. This indicates that divergent initial processes that lead to ARs and LRs may converge on a similar mechanism for primordium development. Furthermore, we demonstrated that when plants are grown in soil or upon wounding on medium, the primary root is able to produce both WOX11-mediated and non-WOX11-mediated roots. The discovery of WOX11-mediated root-derived roots reveals a previously uncharacterized pathway that confers plasticity during the generation of root system architecture in response to different inductive cues.Lateral roots (LRs), which originate from the growing root, and adventitious roots (ARs), which are formed from non-root organs, are the main contributors to the post-embryonic root system in  Summary: Root system development can respond flexibly to developmental and environmental cues by utilizing WOX11-mediated and non-WOX11-mediated pathways, which converge on a common mechanism for primordium development involving LBD16. Arabidopsis thaliana, LRs are initiated from the xylem-pole pericycle cells of a growing root in a developmentally and periodically organized pattern  and adventitious roots (ARs) . In Arab pattern , 2016, w pattern . HoweverThe cellular and molecular processes key to LR formation from the primary root grown vertically on medium have been extensively investigated . GeneralWUSCHEL-RELATED HOMEOBOX11 (WOX11) is required for de novo regeneration of ARs from leaf explants , stem explants  and hypocotyls . Detached leaf and stem explants generate ARs within 8 and 6 days after culture (DAC) on B5 medium, respectively . All plants and explants were grown and cultured in vitro on medium.To analyze the role of ectively . We alsoWOX11 in these rooting systems using two transgenic constructs, pro:WOX1135S and pro:WOX11-SRDX35S, to enhance and block WOX11 activity, respectively , AR formation was greatly enhanced in leaf explants, stem explants and hypocotyls of pro:WOX1135S plants , whereas AR production was severely inhibited in pro:WOX11-SRDX35S plants . However, compared with Col-0, no defects in LR density in pro:WOX11-SRDX35S or enhanced LR density in pro:WOX1135S were observed , although pro:WOX11-SRDX35S had a shorter primary root than Col-0 . Instead, LR density in pro:WOX1135S plants was reduced . This response might be caused by a feedback process due to enhanced adventitious rooting of hypocotyls induced by WOX11 overexpression .We examined the role of ectively . Compareobserved  . WOX11 was induced in all examined AR-competent tissues during AR formation . A detailed analysis using leaf explants showed that WOX11 was initially induced in AR founder cells within 2 DAC   as it developed   . Expressig. S4L) .Fig. S4D-F). More specifically, WOX11 expression was absent in xylem-pole pericycle cells , LR founder cells  and LRP . Instead, expression was detected in protoxylem cells in the primary root meristematic region  . Therefore, LR formation from the primary root grown vertically on medium is controlled by the non-WOX11-mediated pathway.In contrast, GUS signal was not detectable during LR formation (ig. S4E) . In addiWOX11 was also involved in light control of adventitious rooting from hypocotyls . We tested WOX11 expression patterns in leaf explants and primary roots by culturing them on different medium, and the results showed that change of the medium had little effect on WOX11 expression .In addition, we performed experiments in different growth conditions and confirmed that pocotyls  . However, in natural conditions, plants are grown in soil. Therefore, we tested whether the root system grown in soil conditions is able to produce WOX11-mediated roots. Strikingly, in pro:GUSWOX11 plants grown in soil, GUS signal was detected in many regions of the root system . In addition, secondary, tertiary and quaternary root numbers were all reduced in the root system of pro:WOX11-SRDX35S seedlings grown in soil compared with those of Col-0 , the density of the secondary roots derived from the primary roots was also reduced in pro:WOX11-SRDX35S seedlings grown in soil compared with those of Col-0 . In wox11-2 wox12-1 seedlings grown in soil, the number of quaternary roots was reduced compared with those of Col-0 . Taken together, these data suggest that in soil conditions, WOX11-mediated root formation does occur in the root system, which may reflect a response to environmental signals in soil.LR initiation from wild-type seedlings grown vertically on half-strength Murashige and Skoog (1/2 MS) medium or some other media usually did not display a of Col-0 B-D. Althof Col-0 D. In 35SWOX11-mediated root formation. The primary root of 12-day-old pro:GUSWOX11 seedlings vertically grown on 1/2 MS medium was cut at the midpoint to mimic damage of roots. GUS signals could be detected at the excision site within 1 day after excision (DAE) , suggesting that WOX11-mediated root initiation might occur upon excision of the wild-type primary root tip.When plants are grown in soil, they may encounter different nutrition conditions, different types of stresses, damage of roots, and interactions with microbe and insects. We investigated whether damage of the primary root is able to induce WOX11-mediated roots, i.e. ARF7/19-mediated LRs, we performed experiments using the arf7-1 arf19-1 double mutant, which is defective in LR initiation when plants are grown vertically on 1/2 MS medium . Here, we used several approaches to test whether WOX11 is able to regulate LBD16 directly during AR formation from leaf explants.Genetic data showed that explants     Fig. 7ATTAATGG) , was ablATTAATGG LBD16 expression was upregulated in leaf explants carrying the 3×FLAG-WOX11-GR construct (LBD16 remained when treated with DEX together with cycloheximide (CHX), an inhibitor of protein synthesis, indicating the direct regulation of LBD16 by WOX11 . GUS staining of pro:LBD16-GUSLBD16 was induced during ARP initiation in leaf explants at 4 DAC  when plants are grown in soil or when the primary root is damaged . A higher auxin level in soil  is likely to lead to higher WOX11 expression level in soil. Analysis using BAR-Tools (http://www.bar.utoronto.ca/) . The arf7-1 arf19-1 double mutant and the WOX11 and LBD16 marker lines could provide an experimental system for the effects of inductive cues on root branching. Future analysis of the inductive cues will provide a more comprehensive understanding of how root system architecture is established and how developmental plasticity is employed in an effort to optimize response to a variable environment.We observed that the auxin response is different in the primary roots of plants grown in soil and vertically on 1/2 MS medium, suggesting that different environmental cues may cause different auxin responses in roots (nto.ca/)  suggestst system . In addilbd16-2  were described previously . The soil is a 1:1:3 mixture of vermiculite: perlite: peat soil containing 1/2 MS liquid solution without sucrose. Plants were grown in soil in 20-cm-high pots. All plants and explants were grown and cultured at 22°C.GUS staining was carried out based on our protocol . Microscopy and differential interference contrast (DIC) observations were performed as previously described  using a Nikon Eclipse 80i microscope.ACTIN genes. Anti-FLAG antibody  was used in the ChIP analysis. The primers used for real-time PCR are listed in Table S1.ChIP and qRT-PCR were performed as previously described . The qRT


RPeL27, RPeL41, and RPeL43) between cell lines derived from tumor and normal nasopharyngeal epithelium. However, the results therein were based on a semiquantitative assay, thus preliminary in nature. Herein, we provide findings of a deeper analysis of these three genes in the context to nasopharyngeal carcinoma (NPC) tumorigenesis. Their expression patterns were analyzed in a more quantitative manner at transcript level. Their protein expression levels were also investigated. We showed results that are contrary to previous report. Rather than downregulation, these genes were significantly overexpressed in NPC cell lines compared to normal control at both transcript and protein levels. Nevertheless, their association with NPC has been established. Immunoprecipitation pulldown assays indicate the plausible interaction of either RPeL27 or RPeL43 with POTEE/TUBA1A and ACTB/ACTBL2 complexes. In addition, RPeL43 is shown to bind with MRAS and EIF2S1 proteins in a NPC cell line (HK1). Our findings support RPeL27, RPeL41, and RPeL43 as potential markers of NPC and provide insights into the interaction targets of RPeL27 and RPeL43 proteins.Apart from their canonical role in ribosome biogenesis, there is increasing evidence of ribosomal protein genes' involvement in various cancers. A previous study by us revealed significant differential expression of three ribosomal protein genes ( Ribosomal proteins (RPs) are primarily known for their functions in ribosome biogenesis and play a central role in translational processes. In fact, the highly coordinated processes of ribosome biogenesis are also tightly connected to events of cellular growth and development. Dysregulation in these processes could relate to occurrence of diseases that include cancers. It is also an established fact that the phenotypic effects of RP genes extend beyond their canonical ribosomal involvement into extraribosomal functions such as DNA replication, transcription, DNA repair, DNA splicing and modification, and apoptosis . In partNasopharyngeal carcinoma (NPC), a malignancy arising from epithelial cells of the nasopharynx, is a cancer that has been extensively studied with respect to genetic susceptibility and involvement. Early evidence of RP genes involvement in NPC was limited to RPeS26 (RPS26), RPeS27 (RPS27), RPuS19 RPS15), RPeL27 (RPL27), RPeL43 (RPL37a), and RPeL41 (RPL41) , RPeL27  nullifieMolecular pathways or signaling events pertaining to carcinogenesis of NPC, hitherto, cannot categorically include the involvement of RPs. Therefore, to establish RPs as among the factors associated with NPC tumorigenesis, a reevaluation of their expression patterns using more quantitative assay is warranted. This paper reports the reanalysis of expressed transcript level using quantitative RT-PCR strategy of RPeL27, RPeL43, and RPeL41 in NPC cell lines in comparison to normal cell line derived from noncancerous nasopharyngeal epithelium. The upregulated trend of two of these three genes was further substantiated at the protein level, thus validating their association with NPC. Further study on protein-protein interaction reveals plausible binding of two of these three RPs with protein complexes of the cell cytoskeleton, while one of the RPs was shown to possibly interact with two types of oncoprotein.Nasopharyngeal carcinoma cell lines used were SUNE1, HONE1, HK1, and TW01, and nonmalignant nasopharyngeal epithelial cell line was NP69 [−ΔΔCt method.Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. First-strand cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen) by standard method. Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed in three technical replicates with two biological replicates using QuantiFast Probe (Qiagen) on a Rotor-Gene 6000 Rotary Analyzer (Qiagen) and monitored with Rotor-Gene 6000 software version 2.3.3 (Qiagen). The primer-probe pairs used targeted RPeL27, RPeL41, and RPeL43 genes. Beta actin (ACTB) was used as endogenous control for gene expression normalization. Nontemplate reactions were included as negative controls. The fold changes were calculated by using 2Whole cell protein lysates were prepared using RIPA lysis buffer containing protease inhibitor (Roche Applied Sciences). Proteins were separated on 12.5% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (Milipore). After blocking with bovine serum albumin, the membranes were blotted with the primary antibodies for RPeL27 or RPeL43 (Abcam), followed by secondary anti-donkey, rabbit secondary antibody (Abcam). ACTB was used as protein loading control. Analysis was not performed for RPeL41 due to lack of commercially available primary antibody. Protein bands were visualized using enhanced chemiluminescent (eCL) substrate (Promega). Images were captured using ImageQuant TL instrument  and band intensity was read using ImageQuant TL software.Whole cell protein lysate extracted from a NPC cell line (HK1) was incubated with RPeL27 or RPeL43 primary antibody immobilized onto Dynabeads Protein G (Life Technologies) for 10 minutes with rotation at room temperature. The beads were vortexed for 30 seconds, and the vial was placed on DynaMag Magnet (Life Technologies) to separate them from the solution. Following removal of supernatant, sample buffer was added to the vial and this mixture was boiled for 10 minutes. The resulting immunoprecipitates were resolved on SDS-PAGE and stained with Coomassie Blue (Bio-Rad). Protein bands of interest were excised and sent to a service provider for protein identification. Identification analysis was via the Electrospray Ionization-Quadrupole-Time-of-Flight (ESI-QUAD-TOF) method using the Agilent 1260 Infinity HPLC system coupled to Agilent 6540 mass spectrometer (Agilent) and the Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) method using the 5800 Protein Analyzer (AB Sciex). Identities of proteins were extracted using Mascot software (Matrix Science) on Swiss-Prot database. The search parameters utilized trypsin as the proteolytic enzyme with one missed cleavage permitted and the carbamidomethylation of cysteines and oxidation of methionines as variable modifications. Mass tolerance was 50 ppm and 0.4 Da for precursor and fragment ions, respectively.p = 0.044 and 0.011) than in the normal nasopharyngeal epithelial cell line, NP69; and RPeL43 mRNA in SUNE1 and HONE1 cell lines was 2.23- (p = 0.009) and 1.04-fold (p = 0.012) higher, respectively, compared to NP69 (p = 0.013) and 2.05-fold in HONE1 (p = 0.028). However, the fold difference of the three RP genes' mRNA in HK1 and TW01 cell lines is not statistically significant, albeit large differential expression of all three RP genes between HK1 and NP69 cell lines  show overexpression in all NPC cell lines compared to normal nasopharyngeal epithelial cells, while our Western Blot analysis reveals upregulated protein level of RPeL27 and RPeL43 in NPC cell lines relative to a normal control . Transcr to NP69 . This ovll lines . In termll lines .p < 0.05), 13.05 (p < 0.01), and 27.67 (p < 0.05) in HONE1, HK1, and TW01 cell lines, respectively, compared to NP69 cell line. Meanwhile, RPL37a protein level was 3.71 and 7.76 (p < 0.05) in HONE1 and HK1 cell line, respectively. In both protein and transcript levels, RPL27 showed overall higher expression across the NPC cell lines compared to RPL37a. ACTB (Accession NM 001101) was used as reference gene and protein loading control to normalize the expression.Western Blot analysis shows upregulated protein levels of RPeL27 and RPeL43 in the NPC cell lines of HONE1, HK1, and TW01 compared to NP69 Figures . RPeL27 Based on ESI-QUAD-TOF mass spectrum analysis, we identified Muscle Rat Sarcoma Homolog (MRAS) and Eukaryotic Initiation Factor 2 Subunit 1 (EIF2S1) proteins as coimmunoprecipitates of the RPeL43 protein . The preOur results on the significantly upregulated levels of RPeL27, RPeL43, and RPeL41 transcripts in NPC cell lines compared to cultured normal NP cells warrant further discussion. While this expression phenomenon reinforced the fact that they are likely candidates of NPC-associated RP factors, their expression behaviors contradict our previous report [ RPeL27 is upregulated in cells/tissues of sulforaphane treated-breast cancers [ RPeL43 is upregulated in cases of astrocytomas [The overexpression trends of these three RP genes are not uncommon in cancers. For instance, cancers , gastric cancers , hepatoc cancers , and met cancers , while Rocytomas , head anocytomas , 16, andocytomas . The RPeocytomas . Our finIn terms of cell types, significantly higher transcript level of the three RP genes is observed for cell lines derived from the more common type of NPC tissues (Type 2a) comprising nonkeratinized poorly differentiated cells (SUNE1 and HONE1). Despite prevailing overexpression, statistical significance is not apparent in the case of cell lines from well-differentiated NPC cells of either keratinized (TW01) or nonkeratinized (HK1) characteristics. Protein levels of RPeL27 are significantly higher in the three types of cell lines, namely, the nonkeratinized poorly differentiated (SUNE1/Type 2a-NPC), nonkeratinized well-differentiated (HK1/Type 2b-NPC), and keratinized well-differentiated (TW01/Type 1-NPC) cell types. Higher levels of RPeL43 proteins are statistically significant in all cell types except for keratinized well-differentiated ones (TWO1). Taken together, it is clear that this subset of RP genes is most consistently overexpressed in cells from Type 2a-NPC tissues, hence conferring them possible biomarkers for detection and/or progression of this type of NPC. Our findings here provide the first empirical evidence to support this conclusion. Nevertheless, we have yet to further investigate all these observations or prove these inferences in NPC tissues.One of the proteins we found interacting with RPeL43 is EIF2S1. This association is not a surprise bearing in mind that EIF2S1 primarily functions as a translational initiation factor, and its phosphorylation has been known to inhibit protein synthesis . In leukAnother protein identified to be in complex with RPeL43 is MRAS. As activator of other protooncogenes such as Rapidly Accelerated Fibrosarcoma (RAF) kinases , Phosphoγ) colocalizes with actin beneath the cell membrane [For the heavier molecules (POTEE-TUBA1A and ACTB-ACTBL2 complexes) that coimmunoprecipitated with RPeL27 or RPeL43, precise mode of interaction (one-to-one binding) cannot be discerned via our analysis. Each RP's interaction with either one of the complexes could be due to protein aggregation in the assay or actual tertiary structures in the natural state. Under normal circumstances, POTEE  has been explained  and that RPeL27, RPeL41, and RPeL43 as NPC-associated ribosomal protein genes as far as cell line system is concerned. We provide preliminary knowledge on the development of novel markers for early diagnosis and/or prognosis of the nasopharyngeal cancer disease. In addition, we provide early evidence suggesting possible roles of RPeL27 and RPeL43 in the MAPK/ERK pathway.This study verified


Osteosarcoma is an orphan disease for which little improvement in survival has been made since the late 1980s. New drug discovery for orphan diseases is limited by the cost and time it takes to develop new drugs. Repurposing already approved FDA-drugs can help overcome this limitation. Another limitation of cancer drug discovery is the lack of preclinical models that accurately recapitulate what occurs in humans. For OS using dogs as a model can minimize this limitation as OS in canines develops spontaneously, is locally invasive and metastasizes to the lungs as it does in humans.in vitro assays and an in vivo murine model of OS.In our present work we used high-throughput screens to identify drugs from a library of 2,286 FDA-approved drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. The identified lead compound was then tested for synergy with 7 other drugs that have demonstrated activity against OS. These results were confirmed with in vitro combination drug screens. Auranofin showed synergistic effects with vorinostat and rapamycin on OS viability and apoptosis induction. Auranofin demonstrated single-agent growth inhibition in both human and canine OS xenografts, and cooperative growth inhibition was observed in combination with rapamycin or vorinostat. There was a significant decrease in Ki67-positive cells and an increase in cleaved caspase-3 levels in tumor tissues treated with a combination of auranofin and vorinostat or rapamycin.We identified 13 drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. Auranofin was selected for further Auranofin, alone or in combination with rapamycin or vorinostat, may be useful new treatment strategies for OS. Future studies may evaluate the efficacy of auranofin in dogs with OS as a prelude to human clinical evaluation. Osteosarcoma (OS) represents the most common malignant bone tumor with a bimodal peak in adolescents and in those greater than 60 years of age . OverallOS is classified as an orphan disease as it affects only approximately 800 patients per year in the United States. The cost of new drug discovery for orphan diseases is often prohibitive for pharmaceutical companies . One optAs with drug discovery, the lack of preclinical models that accurately recreate what occurs in humans and serve as effective tools in predicting therapeutic response in cancer patients is a challenge for investigators pursuing drug repurposing . In the ®), a FDA-approved oral agent for the treatment of rheumatoid arthritis, was selected for testing its efficacy to suppress OS growth in tissue culture and nude mice, as well as for showing synergistic effects with agents potentially effective in OS therapy. Intriguingly, auranofin had cooperative effects with vorinostat and rapamycin. Our study will advance to a future proof of principle study in canine OS patients and furthermore serve as the rationale for advancing auranofin to human clinical proof of concept studies.Here, we employed a comparative oncology approach to drug repurposing in OS. We evaluated an extensive library of FDA-approved drugs for anticancer activity in validated human and canine OS cell lines in parallel. From these studies, auranofin (Ridaurawt), human osteoblast hFOB 1.19 (p53wt), human OS KHOS/NP (p53R156P), Saos2-LM7 (p53null), MG-63 (p53null), and mouse fibroblasts NIH/3T3 (p53wt) were obtained from ATCC, human osteoblasts NHOst (p53wt) were obtained from Lonza, canine OS Abrams , and D17 (p53wt) were obtained from Douglas H. Thamm . All cell lines used were authenticated 1/m1 + (D)2/(Dm)2[fa/1-fa]1/m2, where CI . The slopes generated by Calcusyn were used to generate normalized isobologram to identify combination data points that fall in the lower left (synergism), on the hypotenuse (additive) or on upper right of the line (antagonism).The growth inhibition of each drug alone was first used to identify Highest Single Agent (HSA). GraphPad Prism software  was used to characterize the drug-drug interaction using Bliss independence (combination effect is independent from the action of second agent: Fa+Fb-FaFb), and Chou-Talalay-based Calcusyn software was used to generate concentration-response curves, median-effect plots and a normalized isobolograms. The median effect plots linearize all concentration-effect curves that follow mass action law principle and were calculated from equation: fa/fu = (D/Dm)This assay was performed to further validate the effects of auranofin on inhibiting proliferation of multiple OS cell lines with minimal effects on that of non-tumor cells. Cells were seeded onto 6-well plates . Twenty-four hours after seeding, cells were treated with 2 different concentrations of DMSO (control) or auranofin. Live cell numbers were counted at days 2, 4, 6, 8, and 10, following trypan-blue staining.Western blotting for PCNA, cleaved caspase-3 and vinculin or GAPDH (control) in KHOS/NP and Abrams cell lines was performed in order to examine the effects of auranofin and its combination with vorinostat and rapamycin on their protein expression. KHOS/NP and Abrams cell lines were chosen due to their aggressive behaviors and abilities to cause metastatic disease. Both cell lines contain mutations in the p53 gene , 19. CelIn order to examine the effects of auranofin and its combination with vorinostat and rapamycin on cell cycle profiles of OS cells, we performed PI-staining and flow cytometry analysis. Cells were fixed overnight with 70% ethanol at -20°C and stained with PI solution (Life Technologies) in the presence of 62 μg/ml of RNase A, followed by flow cytometric analysis using BD Accuri flow cytometer . Data was analyzed using the FlowJo V10 software .in vivo OS growth in nude mice. All mice were purchased from Envigo and were maintained under specific-pathogen-free conditions with regular diet. All experimental procedures were conducted in accordance with the institutional animal welfare guidelines of the University of Kansas Medical Center and we obtained approval from the University of Kansas Medical Center Institutional Animal Care and Use Committee (IAUCUC #2013–2167). Cells (1X106) were subcutaneously injected into 6-week-old female nude-Foxn1nu mice. During injections, mice were gently held and monitored closely for signs of pain or distress. When these signs were noted, the mice were released and the procedure was stopped. When tumors became 3mm in diameter, DMSO, auranofin (0.1 mg/kg), vorinostat (2.5 mg/kg), and rapamycin (0.1 mg/kg), as well as the combination of auranofin with vorinostat or rapamycin were intraperitoneally injected 5 days per week for 3 weeks. Tumor sizes were measured three-dimensionally twice per week during 21 days [To perform proof-of-principle studies toward application to canine and human OS clinical investigation, we examined effects of auranofin and its combination with vorinostat or rapamycin on  IAUCUC #13–2167. After 21 days of treatment with DMSO, auranofin (0.1 mg/kg), vorinostat (2.5 mg/kg), and rapamycin (0.1 mg/kg), as well as the combination of auranofin with vorinostat or rapamycin the tumor tissues were harvested, sectioned at 4 μm, and subjected to IHC by standard procedures. Briefly, sections from the aforementioned tumor tissues were deparaffinized in xylene, rehydrated in grades of alcohol, rinsed in tap water and blocked with 0.3% hydrogen peroxide for 30 min. Antigen retrieval was performed in a steamer with sodium citrate buffer  for 20 min. After blocking in 2.5% normal horse serum for 30 min, sections were incubated with anti-Ki67 , and anti-cleaved caspase-3  for 30 min at room temperature. After washing in PBS, sections were incubated in biotinylated secondary antibody for 30 min. The signal was detected using the Vectastain Elite ABC kit (Vector Laboratories). Pre-immune serum and normal rabbit IgG  were used as negative controls.t-tests with GraphPad Prism 5 . Statistical significance was set at p < 0.05, unless otherwise stated in the text.The differences in cell proliferation, percentages of sub-G0/G1 population, and tumor growth between different treatments were analyzed by two-tailed Student’s 50 values in human and canine OS cell lines (®) demonstrated an in vitro IC50 value ranging from 0.7 to 2.4 μM in all of OS cell lines tested (in vitro combination drug screens based on the following observations: 1) our previous study evaluating auranofin in chronic lymphocytic leukemia patients (NCT01419691) which demonstrates mechanisms of action relevant to the treatment of OS [in vitro synergistic anticancer activity in combination with other pharmacologic classes of anticancer agents [® drug product could be readily administered to human and canine OS patients.To identify whether a currently marketed drug contained within our compound library of 2,286 FDA-approved drugs could suppress growth of human and canine OS cells, we performed high-throughput cell viability assays using 2 human (KHOS/NP and MG-63) and 2 canine (D17 and Abrams) OS cell lines, as well as hFOB human osteoblast cell line as a control . Forty dll lines , Table 1s tested . We selent of OS , 2) in vr agents , 3) the 50 values). Auranofin again showed specificity on OS cell lines to suppress their growth, compared with non-transformed fibroblast cell lines  and two non-transformed mouse and human fibroblast cell lines  with 0.2 μM or 0.5 μM of auranofin  for 24 hours [in vitro.To address the potential mechanism by which auranofin shows cooperative effects on viability of OS cells with vorinostat or rapamycin, we performed propidium-iodide (PI) staining and flow cytometry analyses following treatment of two metastatic OS cell lines KHOS/NP and Abrams with low concentrations (0.5 μM of vorinostat or 1 μM of rapamycin) of these drugs in the absence or presence of 1 μM of auranofin  and/or vorinostat VST, (2.5 mg/kg) or rapamycin  5 days per week for 3 weeks. Tumor sizes were measured twice per week. Combinations of auranofin with either vorinostat or rapamycin significantly suppressed tumor growth of KHOS/NP cells as compared with single treatments of these drugs (in vivo OS growth.We next attempted to examine combinatory effects of these drugs on se drugs . Similarse drugs . During se drugs . These r50 that is achievable in murine models and can be translated into an IC50 that is achievable in humans. Auranofin (Ridaura ®) is an oral gold complex that is FDA-approved to treat rheumatoid arthritis [In this study, we demonstrate auranofin’s ability to inhibit OS cell growth at an ICrthritis . It has rthritis , Ewing srthritis , chronicrthritis , Hodgkinrthritis , and ovarthritis . Auranofrthritis , 28, 29.rthritis , 31. Thirthritis , and henin vitro, inhibit growth of human OS cells, and induce cell cycle arrest both in vitro and in mouse models [et al. [One of the major challenges for successfully treating cancer patients is cancer’s resistance to chemotherapy which can be present at time of initial diagnosis or develop over time with treatment . One waye models –38. Rapae models . Inhibite models . mTOR ane models . Penel-P [et al.  report t [et al. . Even thin vivo studies also showed that there was suppression of OS tumor growth with each drug alone, but tumor growth was significantly inhibited when auranofin was given in combination with either vorinostat or rapamycin. The doses of drugs used in this study were less than those administered in previous studies with other cell lines [Our ll lines , 39, 44.The major reason we evaluated FDA-approved drugs in both human and canine OS cell lines was to capitalize on canines as a model for OS. OS in canines demonstrates many similarities to human OS . CaninesS1 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


MYC rearrangements.3 We describe a novel fusion of FOXR1 forkhead box gene, located at 11q23, with a neighboring gene in B-cell lymphoma.Recurrent chromosome 11q23 abnormalities, including focal gains and losses have been described in mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL) and in a subset of high-grade B-cell lymphomas lacking Ribosomal Protein S25 (RPS25) with FOXR1 in the DLBCL cell line U-2932, both genes located at the amplified chromosomal region 11q23 (RPS25 and to the promoter region of FOXR1 (bp −3532).RNAseq and sequencing of cloned PCR products revealed fusion transcripts of 5′ on 11q23 . Genomic4 These differences also affected 11q32, FOXR1 and RPS25 being tetraploid in subclone R1, and triploid in subclone R2 (3n)  . In accoot in R2 . RPS25/Felopment .FOXR1 (formerly FOXN5) expression is restricted to the early stages of embryogenesis.6 Ectopic expression as result of 11q23 intrachromosomal deletion-fusion has hitherto been described in neuroblastoma only.7 In-frame fusions with the 5′ MLL or PAFAH1B2 genes led to overexpression of FOXR1.7 In accordance with the notion that a constitutively expressed 5′ gene (RPS25) might be responsible for the ectopic expression of FOXR1 in B-cell lymphoma also, FOXR1 levels were 1000 × higher in the fusion-positive than in the fusion-negative U-2932 subclone. Expression array analyses showed that the RPS25/FOXR1-positive U-2932 subclone had the highest FOXR1 expression level of 55 B lymphoma cell lines tested, three log-scales higher than average  yielded wild-type signals only restricted to chromosome 11 (not shown), leaving the putative 5′ regulatory gene elusive.Physiological  average . Quantite U-2932 . High-de2  B lymphoma cell lines. Both cell lines showed amplification of the FOXR1 gene. In one cell line, FOXR1 was fused to a constitutively expressed gene on 11q23, suggesting that the interstitial deletion was responsible for activation of FOXR1. Bioinformatic analyses document that the aberrant expression of FOXR1 is rare, but recurrent in B-cell lymphoma (10Santo lymphoma .8, 9, 10FOXR1 fusions, described as candidate oncogenes in neuroblastoma, also occur in B-cell lymphoma. Cell lines U-2932 and CRO-AP3 are presented as models for the functional analysis of FOXR1-mediated cellular events.In conclusion, we show for the first time that


Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor, with a median survival of less than one year. Due to enormous difficulties in the acquisition of DIPG specimens and the sophisticated technique required to perform brainstem orthotopic injection, only a handful of DIPG pre-clinical models are available. In this study, we successfully established eight patient-derived DIPG cell lines, mostly derived from treatment-naïve surgery or biopsy specimens. These patient-derived cell lines can be stably passaged in serum-free neural stem cell media and displayed distinct morphologies, growth rates and chromosome abnormalities. In addition, these cells retained genomic hallmarks identical to original human DIPG tumors. Notably, expression of several neural stem cell lineage markers was observed in DIPG cell lines. Moreover, three out of eight cell lines can form orthotopic tumors in mouse brainstem by stereotactic injection and these tumors faithfully represented the characteristics of human DIPG by magnetic resonance imaging (MRI) and histopathological staining. Taken together, we established DIPG pre-clinical models resembling human DIPG and they provided a valuable resource for future biological and therapeutic studies. Diffuse intrinsic pontine glioma (DIPG) affects approximately 200-300 children in the U.S. each year. The majority of patients are diagnosed between five and ten years old, with a median survival of less than one year . The staTumor models play a vital role in discovering more effective DIPG treatment methods. They provide us with insights into the biological characteristics of glioma and enable us to test novel therapeutic strategies. Given the genetic and micro-environmental distinctions between DIPG and supratentorial glioma, establishing a specific DIPG pre-clinical model is critical. However, due to scarcity of human DIPG specimens and the sophisticated technique required to perform brainstem orthotopic injection, the development of pre-clinical DIPG models has fallen far behind that of supratentorial glioma models.DIPG pre-clinical models began with allograft models generated from murine glioma cell lines, such as F98 and C6, resulted in the finding that tumors could be formed within the brainstem –11. Humain vitro culture gave rise to tumors consisting of human cells [in vitro and/or in vivo tests for DIPG-targeted therapies were conducted based on these models [Monje et al. developed patient-derived DIPG cell lines and orthotopic xenografts using autopsy specimens . Interesan cells . One cavan cells –20. Notaan cells . Severale models , 23.in vitro and in vivo [Other than patient-derived models, DIPG genetically engineered mouse models (GEMMs) using a replication-competent avian sarcoma-leucosis virus long-terminal repeat with splice acceptor (RCAS)/tumor virus A (TVA) modeling system was also reported . Funato  in vivo . GEMMs aDespite several groups showed the possibility of DIPG autopsy and the feasibility of DIPG biopsy aiming to acquire sufficient specimens for conducting further research, DIPG pre-clinical resources are still extremely limited compared to supratentorial ones, especially the cohort of patient-derived cell lines and xenograft models following the same protocol. In this study, we established eight DIPG cell lines from treatment-naïve specimens. These cells demonstrated variations in morphology, proliferation capacity and chromosome abnormality. Importantly, these cells retained gene mutations from original DIPG tumors and expressed several neural stem cell markers. With these patient-derived cell lines, brainstem orthotopic xenografts were successfully established and their imaging and pathological features were confirmed by MRIs and histopathological staining.Tumor tissues were obtained from eight DIPG patients. The average age of these patients at diagnosis was 6.25 years old. Two patients were male and the other six were female. Five out of eight tumor tissues were obtained from surgery and the other three were from MRI-guided stereotactic biopsy. The histopathologic diagnoses of these tumor samples were singular , but all of them were high-grade gliomas (WHO III and WHO IV). Except for TT11111, who previously received radiotherapy, all other patients were treatment-naïve. The MRI scans of these patients demonstrated infiltrative tumors in pons and the invasion to midbrain, medulla oblongata, and cerebellum . The protocol was approved by the human research ethics committee of Beijing Tiantan Hospital and written informed consent was obtained from the subjects’ parents. After the digestion process, dissociated single-cell suspensions were cultured in Poly-L-ornithine (PLO)/Laminin-coated plates with serum-free neural stem cell medium. We observed that only a sub-population of cells were able to survive and proliferate after initial plating and these cells formed tight small clusters of cells at the first passage. These small aggregates then spread to become more flattened clones. After three to five passages, these cells were free of cell debris and showing as monolayer cells with homogenous morphology and division rate for each cell line. In total, 25 samples were harvested, digested, and cultured and 8 samples could be expanded for more than 20 passages, maintaining stable growth rates and morphology. In these 25 samples, 14 of them were high grade gliomas and 11 of them were low grade gliomas. The success rate of high grade group (8/14) was significantly higher than that of low grade group (0/11). This result indicated that tumor grade contributed the most in determining whether brainstem glioma cell lines can be established successfully. We also observed efficient recovery of each cell line following freezing and thawing. Cell lines derived from different tumor samples displayed different morphologies, indicative of heterogeneity of original brainstem gliomas. These diverse cell morphologies can be roughly categorized as: (1) flattened and/or round, (2) bipolar with long processes, or (3) multipolar Figure . MoreoveP<0.05) between 8 DIPG cell lines in each marker. Thus, DIPG cells we established showed broad molecular similarities to normal neural progenitor cells (NPCs) or oligodendrocyte precursor cells (OPCs).To determine whether the glioma-derived cells had progenitor cell-like characteristics, we analyzed the expression of several neural stem cell lineage markers on these DIPG cells. By immunocytochemistry we found that most of the cells were positive for Nestin, a neural stem cell marker . InteresH3F3A-K27M mutation, the most frequent mutation identified in DIPGs. In addition, we identified five cell lines with TP53 mutations , two cell line with PPM1D mutation (TT10630 and TT10714) and one cell line with wild type for both PPM1D and TP53 (TT11111). Consistent with a previous study [PPM1D and TP53 mutations were mutually exclusive. Interestingly, one line harbored a TERT promoter mutation. No mutation in HIST1H3B, ACVR1, PIK3CA, PIK3R1 and IDH1 was discovered in eight cell lines. Most importantly, all genetic status in cell lines matched their original tumor tissues . Mouse MRI was performed on a 7.0-T machine with axial, sagittal and coronal T2-weighted sequences every 2 weeks or whenever a symptom was found. Three out of eight cell lines  formed tumors within the brainstem area, on 172, 186, and 155 average days after injection, respectively Figure . MRIs dein vitro cultured cells was relatively long, we took another strategy by propagating cells in subcutaneous xenografts prior to orthotopic injection. While all eight cell lines were injected into the flanks of immunocompromised mice, only TT10603 and TT10630 cells generated subcutaneous tumors. The TT10603 subcutaneous tumor was then digested and injected into the mouse brainstem. Interestingly, the mice received serial transplantation displayed symptoms and MRI characteristics much earlier than the mice with direct orthotopic injection. The third and fourth serial transplantation of the TT10603 cell line took only 67 and 40 average days, respectively, to generate orthotopic tumors, representing a significantly decreased latency compared to direct orthotopic injection  were injected into brainstem after subcutaneous propagation. Mice in all the groups formed tumors within 70 days, verifying that orthotopic xenografts can be initiated even by a small amount of DIPG cells  and xenografts from serial transplantations (TT10603) retained the H3F3A-K27M mutation of their original tumors. Immunohistochemistry staining of H3K27me3 demonstrated that orthotopic xenografts from both direct orthotopic injection and serial transplantation lost the trimethylation at position 27 of histone H3, indicating that the models retained similar characters as the H3F3A-K27M mutant human DIPG  a patient-derived origin retaining the characteristic of human DIPG; (2) a specific brainstem microenvironment created by precise orthotopic injection; (3) a cohort of resource which can be explicitly stratified in order to test novel therapeutic strategies; and (4) a stability and reproducibility which enables the model to be repeated according to a standard protocol. The objective of our study was approaching to these criteria as closely as possible.In our study, several strategies were applied to assure the success of establishing cell lines resembled the characteristics of human DIPG. First, we chose treatment-naïve patients for a better preservation of original human DIPGs. Secondly, we used serum-free culture media with specified growth factors to avoid genetic alterations due to the presence of fetal bovine serum –31. The H3F3A, TP53, and PPM1D were the most frequently mutant genes in human DIPGs. Our established cell line maintained all these important genetic alterations from original tumors, which provided great opportunities for studying molecular mechanisms of DIPG. H3F3A was considered to be the most frequent and the most defining mutation associated with poor survival in DIPG [TP53, PPM1D, ACVR1 and PIK3R1 were regarded as main driver mutations of DIPG as well [TP53 and PPM1D mutations suggested distinct molecular pathways for tumorigenesis [H3F3A mutation accompanied with TP53 mutation ; (2) H3F3A mutation accompanied with PPM1D mutation ; and (3) H3F3A mutation without TP53 or PPM1D mutations (TT11111). This classification might provide clues on understanding clinical classification of DIPG at molecular level. Furthermore, given that both H3F3A and PPM1D were considered as therapeutic targets for DIPG, our DIPG cell lines not only can be served as great tools to study biological function of DIPG, but also can be used for novel drug screening. Interestingly, TERT promoter mutation at C250T was identified in patient TT10902 in both tumor and cell line, which may help understand the telomerase function in DIPG.Development in cancer genomic sequencing greatly advanced the progression of molecular classification and targeted therapy of brain tumor.  in DIPG , while T as well . The mutigenesis . Our est3 cells was sufficient to form intracranial tumors suggested these cells were highly tumorigenic. Altogether, the expression of stem cell markers, the ability of forming neurosphere and the capacity of forming tumors with a low cell number supported the conclusion that GSLCs existed in DIPG and may be the cause of DIPG's resistance to radiotherapy and chemotherapy.Glioma stem-like cells (GSLCs) display characteristics of self-renewal capacity and tumor-initiating ability , 35. Whiin vitro cultured cells instead of direct injection of fresh human tumor tissues specimens, owing to the result of an interesting study that direct injection of fresh specimens without in vitro culture will lead to murine but not human tumors [in vivo method to monitor the tumorigenesis progress in mice, same as it has been done in clinic for human patients. Together with the retaining of protein markers, the status of H3F3A mutation and the loss of trimethylation at position 27 of histone H3, these orthotopic models mimicked the anatomical, histological and molecular features of human DIPG.The establishment of DIPG cell lines faithfully resembling the characteristics of human DIPG laid the foundation to the successful development of DIPG orthotopic xenograft models. Notably, these xenografts were generated by n tumors . MRI plan tumors . The estin vitro cultured cells adapt to in vivo condition before intracranial injection. Interestingly, tumors from serial transplantation also showed a more extensive infiltration pattern on MRI and higher level of neural stem cell markers, compared to that from direct orthotopic injection and original human specimens. Although the subcutaneous microenvironment was not identical to the CNS microenvironment and might result in overestimation of angiogenesis inhibitors [The long latency of tumor formation by direct orthotopic injection could be a concern for functional and therapeutic research. Passaging as subcutaneous xenograft prior to orthotopic transplantation could maintain the signature of primary glioma cells while facilitating intracranial tumorigenesis–41. In ohibitors , this sein vitro drug screening and in vivo drug testing. Therefore, the establishment of our faithful DIPG cell lines and xenograft models may shed light on future investigations for more effective therapeutic strategies of this devastating disease.In summary, we successfully established sustainable patient-derived DIPG pre-clinical models, including cell lines as well as orthotopic xenografts through direct injection and serial transplantation. These reproducible models will not only help us make insights into the biological characteristic and molecular mechanism of DIPG, but also be valuable tools for Tumor tissues were dissected from patients during surgery and collected in sterile Hibernation media  and tran4 DIPG cells. After 48 hours, cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, then incubated with primary antibody overnight at 4°C. After that, cells were rinsed with PBS and incubated with fluorescence-labeled secondary antibody  for 45min at room temperature. Meanwhile, cells were counterstained with DAPI. After rinsing with PBS, coverslips were mounted on slides with Fluoromount-G (SouthernBiotech). Images were obtained using a Zeiss microscope. Primary antibodies Ki67 , Nestin , GFAP , Olig2  and PDGFRα  were used.Coverslips, coated with PLO/Laminin (3ug/ml), were placed into 24-well plates and seeded with 4 × 10H3F3A , HIST1H3B (K27), TP53 , PPM1D (exon 6), TERT promoter region , ACVR1 , PIK3CA , PIK3R1 (N564) and IDH1 (R132). Sanger sequencing of H3F3A  was performed for xenograft samples. The sequencing was performed by Genotron Health (Bejing) Co. Ltd, .Sanger sequencing of frequently mutant genes in DIPG were performed for all eight established DIPG cell lines and their corresponding human tumors. The gene list includes DIPG cells were seeded and cultivated in 96-well plates  on day 0. Every other day, the plate was added 10μl CCK-8 per well and incubated for 4 hours in the incubator. Then the absorbance was measured at 450nm using a microplate reader. The growth curve was plotted using absorbance values and each point referred to triplicates. Cell doubling time was calculated by the following formula: doubling time=ln(2) · t/ln(N(t)/N(0)) t=10, where n was the cell number and t was the hours in culture.DIPG cells were cultured to about 70% confluency, then treated with colchicine (25ug/ml) for 6h and preheated 0.075mM Kcl for 30 min at 37°C. After that, cells were added with fresh stationary liquid  and centrifuged at 1200 rpm for 10 min. Then the pellets were fixed at room temperature for 10 min and 500ul stationary liquid was added to resuspend them. Cell droplets were applied to clean microslides that had been soaked in 4 to 6°C ice water. The samples were baked in 80°C constant temperature drying oven for 15 min. After Giemsa staining, the microslides were assessed by optical microscopy.5 cells in 5ul media was used to perform the injection under the control of Nanoject III Programmable Nanoliter Injector (Drummond Scientific Company). The coordinate was 1.5mm posterior to lamda, 1.0mm lateral to midline and 4.5mm beneath the skull. Syringe was slowly removed to prevent efflux. A small amount of bone wax was applied to the skull where the needle penetrated, and skin was sutured. After the injection, weight was recorded and mice were checked every 48 hours. Every two weeks or whenever any symptoms were identified, MRI was performed by 7.0-T mouse MRI. Mouse was sacrificed when tumor was confirmed by MRI or nine months after injection.For brainstem injection, NGS mice were performed intraperitoneal injection of chloral hydrate for anesthesia and positioned into a digital stereotaxic frame (Stoelting). A 1.5cm midline skin incision was made over the posterior skull of the animal. Lambda was located as landmark. A small hole was created by puncture with an electric drill. A glass capillary loaded with 106 DIPG cells were injected into the flank of NSG mice, and the tumor volume was measured with caliper every 2-3 days. Once tumor diameter reached 1.5cm, tumors were dissected and digested before being injected into the brainstem of NSG mice.As for serial xenograft, 10MRI experiments were performed in a 7.0-T magnet . A surface coil (2.3 cm × 1.5 cm) was used for brain imaging. Axial, coronal and sagittal T2-weighted images were acquired using a SE sequence .2O2, and antigen retrieval was performed using heated sodium citrate treatment. Afterward, primary antibodies were added to each slide at appropriate dilutions, and the sections incubated with polyperoxidase-labeled secondary antibodies for 25 minutes. The final signals were developed using the 3, 3’-diaminobenzidine substrate (DAB) (MXB Biotechnologies). The sections were analyzed by optical microscopy after counterstaining with hematoxylin.The tissues were fixed in 10% formalin, followed by dehydration in ethanol gradients, permeabilized with xylene and paraffin embedded. Sections with 5μm thickness were deparaffinized with xylene, hydrated with ethanol gradient and stained with hematoxylin and eosin (H&E) (MXB Biotechnologies). Immunohistochemical staining for Nestin , Sox2 , Olig2 , PDGFRα , GFAP  and H3K27me3  was performed. Endogenous peroxidase inactivation was carried out with 3% H


TP53 (80%), PTEN (35%), RB1, and BRCA1 genes [Breast cancer arises from luminal or basal cells lining the mammary duct and has been classified into four subgroups based on gene expression: luminal A, luminal B, HER2 enriched, and basal-like . The majThe McDaniel et al study  furthereSTAT3-specific regulation of gene expression was determined by gene expression changes in TNBC cell lines after STAT3 knockdown. STAT3 binding sites were identified within 50KB of 64% and 39% of genes differentially expressed after STAT3 knockdown in HCC70 and MDA-MB-231, respectively. Twenty-two genes were regulated by STAT3 in both cell lines, but this small signature was not enriched for any biological processes suggesting inherent heterogeneity within the cell lines. However, both cell lines were enriched for genes involved in invasion and migration, and integration of all datasets across five cell lines  revealed significant enrichment in genes involved in invasive processes. STAT3 specific knock down in two of the cell lines impaired invasion, but did not affect viability or proliferation [These data provide a comprehensive characterization of gene regulation by STAT3, and show a role for STAT3 in promoting invasive properties in TNBC. Data from TNBC cell lines and tumors show regulation of genes unique to individual cell lines and common among them all. What's not in common could be important for driving heterogeneity among TNBC tumors, and also begs the question about what other transcription factors are cooperating to drive a STAT3 program. Conversely, it is possible that only the overlapping genes drive TNBC although this overlapping dataset does not have an invasive signature.One area of interest for the future concerns the mechanism of STAT3 activation in the cell lines, which was not determined in this study. While the correlation of binding sites observed in actual tumors was a powerful validation for the findings in cell lines, STAT transcription factors can be regulated by a plethora of upstream ligands, receptors and kinases, and the primary route of STAT activation in the TNBC tumors and cell lines in this study remains unknown. In the cell lines examined, STAT3 activation was, at least in part, due to components of serum in the media. The IL-6/JAK2/STAT3 pathway is required for growth of basal-like breast cancer cells (including MDA-MB-231 and MDA-MB-468) implicating IL-6 and JAK2 as possible regulators [In addition to JAK2/STAT3, p53 also plays a critical role in TNBC. Not surprisingly, p53 alterations occur in all five TNBC cell lines studied, four of which have p53 missense mutations (one has a frame shift mutation). A large body of data indicates that mutant p53 proteins have additional activities that fuel metastatic properties such as invasion and motility . Data frTP53 mutant TNBC, is the potential role for STAT3 in other subtypes of breast cancer that are predominately TP53 wild type. While STAT3 may not be constitutively active in these tumors, it can be strongly activated by cytokines induced in tumor cells (including breast) made senescent by genotoxic chemotherapy [STAT3 binding activity in breast cancer cell lines derived from other subtypes and cultured in serum was not examined. How much of the profile described here is unique to the TNBC cell lines and tumors? One possible extension of these studies that were focused on constitutive STAT3 activation in predominantly otherapy , 9. In vIn sum, McDaniel et al present an unbiased, genomic scale characterization of STAT3 transcriptional regulation in TNBC. These data will be an invaluable resource for future studies examining STAT3 regulation across different tumor types and model systems.