Here, we demonstrated its ability to reduce cell viability, induce apoptosis and cell cycle arrest in lymphoid cell lines such as Jurkat, SKMM1, Derl-2/7, and myeloid cell lines, such as Jurl-MK1. Moreover, we reported a high expression of a Src kinase, Fyn, in these cell lines compared to healthy subjects. This study was a starting point to investigate 4c pyrazolopyrimidine compound as a drug for HMs and Src kinases as its potential molecular targets.Molecular targeted therapies are based upon drugs acting on tumors by interfering with specific targets involved in growth and spread of cancer. Many targeted therapies were approved by Food and Drug Administration as standard treatment, others were introduced into preclinical or clinical studies on hematological malignancies (HMs). The development of drug-resistance in some HMs and the lack of effective treatments in other ones emphasized the need for searching new molecular targets and therapeutic agents. The aim of this study was to evaluate the effects of 4c pyrazolo[3,4- Targeted cancer therapies are “drugs” designed to interfere with specific molecules necessary for tumor growth and progression. These agents are broadly classified in monoclonal antibodies and small molecules. The first are generally directed against antigens expressed on neoplastic cell surface, the second are usually designed to interfere with the enzymatic activity of the target protein. A primary goal of these kind of therapies is to fight cancer cells with more precision while to do less damage to normal cells.The approval of imatinib mesylate for the treatment of chronic myeloid leukemia (CML) and its high success rate supported the extensive efforts to develop novel molecularly targeted therapies for hematologic malignancies . On the A lot of new compounds have been synthesized, investigated, and introduced into pre-clinical and clinical studies in hematological malignancies HMs; . The majThe Src family protein tyrosine kinases (SFKs) are non-receptor intracellular kinases known to have diverse and important regulatory roles in both normal hematopoiesis and leukemogenesis. There are eight members of this family in mammals and they are structurally related, except for the unique domain: Src, Lyn, Hck, Fyn, Yes, Blk, Fgr, and Lck . SFKs red]pyrimidine library of SFKs inhibitors, which entered in ATP binding site and showed antiproliferative and pro-apoptotic action in several tumor types such as CML and in Burkitt lymphoma  cell leukemia and in CML. In particular, it reduced phosphorylation of Fyn kinase in these neoplasms . The study was approved by the Ethics Committee of IRCCS-CROB  and all subjects gave informed consent according to the Declaration of Helsinki. Peripheral blood mononuclear cells from HDs (HD-PBMCs) were isolated by Ficoll-hypaque gradient separation.2.Both human lymphoid and myeloid cell lines were purchased from Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH or from American Type Culture Collection and cultured as follows. Jurkat (acute T cell leukemia), Derl-2 and Derl-7 (T cell lymphoma), NALM-6 and BV173 (acute lymphoblastic leukemia), Jurl-MK1 (CML), NB4 and HL-60 (acute promyelocytic leukemia), Kasumi-1 (acute myeloid leukemia) cell lines were cultured in RPMI 1640  supplemented with 20% fetal bovine serum  and 1% of penicillin-streptomycin . For Derl-2 and Derl-7, 20 ng/ml interleukin-2  was added at the previous indicated medium. OCI-AML3 cell line (acute myeloid leukemia) was cultured in Dulbecco’s Modified Eagle Medium  supplemented with 20% FBS, 1% of pen/strep. SKMM1 cells (multiple myeloma) were cultured in Iscove’s Modified Dulbecco’s Medium  supplemented with 10% FBS, 1% of pen/strep. All cell lines were maintained in incubator at 37°C and 5% COd]pyrimidine compound, given by Lead Discovery Siena s.r.l. (patent: WO2016066755), was dissolved in dimethyl sulfoxide  and diluted in FBS for cell treatments.4c pyrazolopyrimidine compound or with DMSO vehicle control at increasing concentrations (1–15 μM) from 24 to 72 h to verify its effect on cell viability.We treated 3 HD-PBMCs, 6 lymphoid and 5 myeloid cell lines . In the context of lymphoid malignancies , Jurkat, SKMM1 and Derl-2 cell lines showed a wide reduction of cell viability (about -50%) already at 5 μM and it increased at higher concentrations of 4c compound, reaching a reduction of cell viability of 60–70% at 10 μM. This effect was observed at 24 h and remained constant at other time points. Derl-7 and BV-173 showed a dose-dependent viability reduction after treatment with 4c compound with the higher effect at 48 and 72 h, -25 and -60% at 15 μM, respectively. On NALM-6 cell line, we observed a reduction of viability of 40% at 15 μM of 4c compound at all time points.First of all, we observed that 4c compound had negligible effect on HD-PBMCs at 15 μM in a time course, from 24 to 72 h , Jurl-MK1 and NB-4 showed a dose-dependent reduction of viability after 4c compound treatment, especially at 48 and 72 h.In the context of myeloid malignancies . For lymphoid neoplasms, Jurkat cells showed EC50 around 4 μM at 24, 48, and 72 h; SKMM1showed EC50 = 7.7 μM at 24 h which increased at 48 and 72 h; Derl-2, Derl-7 and BV-173 showed a decrease of EC50 values over time. For NALM-6 EC50 value was much greater than the highest concentration used. For myeloid malignancies, Jurl-MK1 showed EC50 > 15 μM at 24 h but it considerably decreased at 2.5 μM at 48 and 72 h. The same trend was observed in NB-4 cells, in which EC50 was >15 μM at 24 h and it decreased at 48 and 72 h at 7.2 and 5.9 μM, respectively. HL-60 and OCI-AML3 had EC50 higher than 15 μM at all time points. Kasumi-1 showed EC50 = 15 μM at 48 and 72 h.We calculated 4c compound ECFigure 2). Jurkat cells were treated with 2 μM of 4c compound for 24, 48, and 72 h. After treatment we observed a significant increase of apoptotic cells at all time points respect to their control, 34% at 24 h, 18% at 48 h, and 27% at 72 h (p < 0.01). The treatment with 4 μM of 4c compound for 72 h on Derl-2 and Derl-7 induced an increased apoptosis respect to their control . Jurl-MK1 cells, after incubation with 1 μM of 4c compound for 72 h, showed an increased apoptosis rate respect to control .To further investigate cell death mechanism induced after treatment, we performed apoptosis on Jurkat, Derl-2, Derl-7, and Jurl-MK1 cell lines by cytometric analysis of Annexin V/PI . Jurkat were treated with 2 μM of 4c compound for 24, 48, and 72 h; Derl-2 and Derl-7 with 4 μM of 4c at for 72 h; while Jurl-MK1 with 1 μM of 4c compound for 72 h. Jurkat showed cell cycle arrest in G0/G1 phase in all time points, especially at 24 h, respect to control. Derl-2 displayed a strong arrest in G0/G1 phase, whereas Derl-7 manifested a negligible effect on cell cycle after treatment, respect to control. Jurl-MK1 showed a small increase of G0/G1 phase and a larger raise of percent of cells in S phase.We also performed cell cycle analysis on Jurkat, Derl-2, Derl-7, and Jurl-MK1 cell lines by cytometric analysis of PI .We analyzed Fyn protein expression in 3 HD-PBMCs, in lymphoid and myeloid cell lines by WB analysis. Fyn is over-expressed in lymphoid cell lines respect to HD-PBMCs; in particular, SKMM1 showed the major amount of protein, following by NALM-6, Derl-7, Jurkat, Derl-2, and BV-173. Also all myeloid Jurl-MK1, NB-4, OCI-AML3, and Kasumi-1 cells presented higher Fyn protein expression  chromosome, lacking instead in NALM-6. In the context of myeloid cell lines, we observed an EC50 achievement at concentration ≤15 μM at 48 and 72 h for Jurl-MK1, NB-4, and Kasumi-1 cell lines. Jurl-MK1 response could be comparable to lymphoid BV-173 cells probably due to the presence of Ph chromosome in both cell lines.Interestingly, Jurkat, SKMM1, Derl-2, Derl-7, and BV-173 showed a dose-dependent reduction of cell viability and their ECs TcRγδ- . Derl-2,In general, lymphoid cells seemed to respond better than myeloid ones. In agreement with our data, a lot of recent studies showed that the use of TKIs, especially against Btk, Syk, and Lyn, is a promising new strategy for targeted treatment of B-cell lymphoid malignancies . FurtherWe decided to study in deep the viability reduction in Jurkat, Derl-2, Derl-7, and Jurl-MK1 cell lines and we demonstrated that 4c compound induced apoptosis and cell cycle arrest.Recent studies showed that other small molecules induced cell viability reduction and apoptosis in cell lines used in our study. For example, in vitro enzymatic assay, that 4c compound inhibited different Src kinases, in particular Fyn with IC50 = 0.07 μM (Since we previously observed, by  0.07 μM , we veriFyn, a member of SFKs, has diverse molecular functions, including regulation of cell growth, survival, adhesion, cytoskeletal remodeling, motility, axon guidance, synaptic function, platelet activation, and T cell receptor signaling. It is involved in various aspects of the pathogenesis of different types of cancers as well as drug resistance  so it coIn a rare NK leukemia, we demonstrated that antiproliferative activity of 4c compound was due to Fyn kinase inhibition . RecentlOur preliminary data indicated Fyn as a potential target of 4c compound but cannot be excluded that 4c compound might also act on other Src kinases activated in neoplastic cells. Other experiments are needed to evaluate this hypothesis.d]pyrimidine compound had cytotoxic effect by inducing a reduction of cell viability and a increased apoptosis in different cell lines derived from HMs. Finally, we observed that 4c compound potential target, Fyn kinase, is over-expressed in all cell lines used. But others experiments are necessary in this fields, because this study represents a starting point to better investigate the effect of 4c pyrazolopyrimidine compound and the role of Src kinases in HMs.In summary, in this study we demonstrated that 4c pyrazolo[3,4-All authors participated to the conception of the study; IL, designed the work, planned and performed experiments, performed flow cytometry analysis, analyzed data, drafted paper; AC, designed the work, planned experiments, analyzed data, drafted paper; FL, ST, LD, FD, performed experiments and analyzed data; SS and MB, designed and synthesized chemical compound; GF improved writing paper; LD and PM, designed the work, improved writing paper; all authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The National Cancer Institute-60 (NCI-60) cell lines are among the most widely used models of human cancer. They provide a platform to integrate DNA sequence information, epigenetic data, RNA and protein expression, and pharmacologic susceptibilities in studies of cancer cell biology. Genome-wide studies of the complete panel have included exome sequencing, karyotyping, and copy number analyses but have not targeted repetitive sequences. Interspersed repeats derived from mobile DNAs are a significant source of heritable genetic variation, and insertions of active elements can occur somatically in malignancy.Alu Short INterspersed Element (SINE) insertions in cancer genes in NCI-60 cells. We focused this discovery effort on annotated Cancer Gene Index loci.We used Transposon Insertion Profiling by microarray (TIP-chip) to map Long INterspersed Element-1  and Alu insertion sites, respectively. As expected, these numbers encompass previously known insertions, polymorphisms shared in unrelated tumor cell lines, as well as unique, potentially tumor-specific insertions. We also conducted association analyses relating individual insertions to a variety of cellular phenotypes.We catalogued a total of 749 and 2,100 loci corresponding to candidate LINE-1 and Alu retrotransposons. Our findings also indicate that as large numbers of cancer genomes become available, it will be possible to associate individual transposable element insertion variants with molecular and phenotypic features of these malignancies.These data provide a resource for investigators with interests in specific cancer gene loci or mobile element insertion effects more broadly. Our data underscore that significant genetic variation in cancer genomes is owed to LINE-1 and The online version of this article (doi:10.1186/s13100-016-0078-4) contains supplementary material, which is available to authorized users. Alu insertions in NCI-60 cancer cell lines. This provides a resource for discovering and testing functional consequences of these sequences.Transposable elements are repetitive sequences that comprise much of our DNA. They create both inherited and somatically acquired structural variants. Here, we describe a first generation map of LINE-1 and The National Cancer Institute-60 (NCI-60) cell panel was developed in the 1980s as a tool for pharmacologic screens and has become the most extensively studied collection of human cancers . They haNCI-60 cell genomes have been described by targeted  and wholAlu SINEs  are sources of genetic polymorphisms where both the pre-insertion allele and the insertion allele coexist in human populations. Moreover, LINE-1 sequences are hypomethylated [Interspersed repeats have not been incorporated in these or many other genome-wide surveys. These repetitive sequences are dynamic constituents of human genomes and important sources of structural variation –20. RNA thylated –28 and ethylated , and somthylated , 30–36.It is well established that inherited and acquired mobile DNA insertions can affect gene expression; there is inherent potential for insertions to have effects on tumor biology. However, the large majority occur in intronic or intergenic regions. Strong biases in the distribution of insertion sites or recurrent ‘hotspots’ for insertions arising during tumor development are frequently not obvious, leading to the presumption that most are non-functional ‘passenger mutations’ , 36.not such a tumor-normal comparison study, but rather, one aimed to identify potential functions of mobile DNAs in human cancer cells. Towards this end, we mapped LINE-1 and Alu insertions in the NCI-60 tumor cell panel. We used a method for interspersed repeat mapping, Transposon Insertion Profiling by microarray (TIP-chip), to identify insertion sites. We also use previous characterizations of the cell panel to associate specific insertions with cellular phenotypes.This is AluYa5/8; and AluYb8/9) as previously described [Alu elements incorporated in the reference genome assembly  were used as a quality control metric and to set cut-offs for recognized peaks , which uses vectorette PCR to amplify unknown sequence adjacent to a known primer-binding site Fig. . We survescribed . To focuaks Fig. .Fig. 1MaAlu insertion sites respectively were recognized across the NCI-60 cell panel. These locations were cross-referenced to previously described insertions to define three categories: (i.) reference insertions, which include invariant insertions and insertion polymorphisms incorporated in the reference genome assembly; (ii.) inherited variants either previously described (known polymorphic) or newly discovered, but occurring in multiple, unrelated cell lines (novel polymorphic); and (iii.) novel, ‘singleton’ insertions seen uniquely in one cell line . Density plots for both LINE-1 and Alu show most peaks fall into this last category, particularly for L1Hs, although a biphasic distribution was seen  segregating in human populations  locus was only absent in the MB468 breast cancer cell line, consistent with the homozygous deletion of RB1 reported for this cell line [We found that each cell line had a unique transposable element (TE) insertion profile Fig. . After cell line .In TIP-chip, probe spacing does not resolve insertions to the precise base, and insertion strandedness was not predicted for all peak intervals in this study. Despite these limitations, we identified peak intervals that partially or entirely overlapped exon intervals for further inspection. Partial overlaps were almost entirely attributable to insertions near an exon. We identified 9 insertions within exons, and all were located within gene 3’ untranslated regions (3’ UTRs); none affected protein open reading frames.Alu insertions in “STOP genes”, defined in shRNA screens as suppressors of human mammary epithelial cell proliferation [p = 1.23x10-9)  Fig. . This reset Fig. . AnalysiConsistent with this model, ovarian cancer cell lines showed a preponderance of insertions in genes that are down regulated in ovarian cancers as compared to normal tissue. A random set of genes from the array is shown as a histogram for comparison Fig. . This pap = 1.46x10-4). The latter was assessed using genes frequently mutated in human cancers taken from the Catalogue Of Somatic Mutations In Cancer (COSMIC) database [p = 7.74x10-10)  defined as gene loci recurrently interrupted by insertional mutagens in forward cancer gene screens in mice , 40 (p =database  (p = 7.710) Fig. . We also10) Fig. . We antip-value for each association (S5–25). Direct, local roles for TEs (in cis) were not observed for the majority of correlations. However, COMPARE did reveal three insertions associated with DNA hypermethylation within 30 kb of the insertion site. For example, a polymorphic Alu insertion in the SS18L1  gene locus oriented anti-sense to the transcription of the gene, is associated with increased methylation of nearby CpG sites at the same gene locus (p = 6.67x10-6)  Fig. .Fig. 5TEtrans , and more than 10 insertions were associated with thymidylate synthase activity . To probe relationships between multiple trans associated factors related to a single TE insertion, we performed pathway analyses on sets of genes, each encompassing the TE insertion locus and all RNAs and proteins with associated expression patterns. This yielded more than 250 curated pathways with enrichment p-values less than 10-4, supporting the concept that these are biologically relevant as opposed to spurious associations. All COMPARE results are provided in the , LINE-1 has been more well documented to be active in cancer. Alu insertions account for more inherited polymorphisms. For both types of TEs, the vast majority of catalogued insertions are intronic and intergenic without clear function.Our genomes are filled with highly repetitive DNA sequences derived from TEs. Tailored methods for their detection, including TIP-chip , targeteAlu integrations in human neoplasias, we mapped these variants in the NCI-60 cell panel. NCI-60 is a unique resource for this, encompassing a variety of cancer cell lines that have the advantages of being well studied and readily available. We mapped LINE-1 and Alu insertion positions using a microarray-based approach over a large census of cancer genes. Even as TIP-chip is replaced by sequencing, we expect these data will provide a useful reference.To begin a systematic survey for functionally consequential LINE-1 and AluYa/Yb insertions distributed across the 60 cell lines. These include insertions that are unique to a cell line (‘singleton’) and novel polymorphic insertions (found in unrelated cell lines). Although ‘singletons’ may be enriched for tumor-specific, somatic insertion events, matched non-neoplastic cells for the corresponding patient cases are not available, and therefore we cannot definitively differentiate somatic from inherited variants. Similarly, these cell lines have undergone numerous passages since their creation, and somatic insertion events occurring in culture cannot be clearly recognized. We note a greater proportion of LINE-1 singletons (68 % of LINE-1 loci) than Alu singletons (21 % of Alu loci), consistent with ongoing LINE-1 retrotransposition in vivo or in vitro.TIP-chip across the NCI-60 panel revealed numerous novel candidate TEs, totaling about 500 L1Hs and 1000 We approached the question of TE function by two avenues. We first tested for biases in the distribution of insertions with respect to known gene sets. We found a preferential accumulation of TE insertions in retained copies of ‘STOP genes’ in breast cancer cell lines; these gene loci function as inhibitors of mammary epithelial cell proliferation. Experimental models suggest that it is advantageous for tumor growth to compromise the function of these genes , and we Alu sequences [We note that the exonizations of intronic LINE-1  and Alu equences  are beincis effects could be seen relating individual TEs with local DNA hypermethylation. We identified three Alu integrations associated with DNA hypermethylation at the insertion site (+/- 30 kb). The most notable is a polymorphic Alu insertion in the first intron of the SS18L1  gene locus associated with CpG hypermethylation at the same locus (p = 3.67x10-6). SS18 and SS18L1 encode transcriptional regulators and are breakpoints in chromosomal translocations in synovial sarcoma [Alu insertion impacts expression of this gene is unknown. So, while it is not clear at this point that SS18L1 methylation is germane to the development of these malignancies, our ability to relate genotype and epigenetics at these sites demonstrate the value of this approach.Our second approach relied on association studies. We used existing data in COMPARE analyses to test for relationships between TE insertion alleles and cellular phenotypes. In the case of DNA methylation only,  sarcoma . These ttrans effects that are difficult to test further. Pathway analyses suggest that many are not random, but reflect recognized, related gene sets. It may be that the indirect effects can be dissected for some insertion alleles; particularly promising may be those at loci of transcriptional regulators with definable target genes [The large majority of statistically significant associations between insertions and cellular phenotypes appeared to involve indirect or et genes .Alu insertion sites in a panel of widely used cancer cell lines, the NCI-60. We expect maps such as these will be a useful resource for experimentalists with interests in how transposable element insertions interact with genes. Our analyses show that insertion sites can be integrated with other data to develop testable hypotheses about the function of mobile DNAs in cancer.In summary, we profiled LINE-1 and The National Cancer Institute-60 (NCI-60) human cancer cells are a group of 60 cell lines representing nine different types of neoplasias composed of 54 individual cancer cases and three pairs of cell lines  with each pair originating from the same patient , 49. TheA genomic tiling micorarrray was designed to cover the NCI Cancer Gene Index (disease list). A total of 6,484 RefSeq gene identifiers were extracted from the. XML file and converted to genomic coordinates corresponding to each transcript unit +/- 10 kb hg19 reference genome assembly . UCSC Table Browser intervals were merged using GALAXY , and proAseI, BspHI, HindIII, and Xbal). Sticky ends were ligated to annealed, partially complementary vectorette oligonucleotide adapters. Each template was aliquoted into 3 separate vectorette PCR reactions for L1Hs, AluYa5/8, and AluYb8/9 mobile DNA families. These were then labeled with Cy3-dUTP for LINE-1 and Cy5-dUTP for Alu and hybridized to Nimblegen genome tiling arrays according to the manufacturer’s instructions. Reference insertions are those incorporated in the Feb. 2009 assembly of the human genome .Five micrograms of genomic DNA of each cell line was digested overnight in parallel reactions using four restriction enzymes  to give genomic coordinates and probe intensities (.gff files). A PERL script removed probes overlapping repeats to reduce noise (RepeatMasking). Nimblescan called peaks using a sliding window threshold. Peaks were ranked by the threshold of the log2 transformed ratio of red (Alu) and green (L1) channels or the reciprocal (settings: percent (p) start = 90, p step = 1, #steps = 76, width of sliding window = 1500 bp, min probes > 4, all probes > 2). The top 5,000 L1 and Alu peaks were kept for evaluation.Among these peaks, recovery of those corresponding to mobile DNA insertions in hg19 (reference insertions) was used as a proxy of assay performance. Reference insertion count was plotted against peaks recognized Fig. . A cut-oPrinciple component analysis (PCA) (R-package) was used to remove batch effect. All insertions were sorted by density across the cell lines and plotted as a matrix. Cell lines lacking high-frequency insertions were assessed for karyotype abnormalities manually.cis effects and were discarded from other associations due to their high false-positive rates. P-values for other insertions were corrected using Bonferroni multiple test correction and plotted using the start position of peak intervals to generate Manhattan plots .Reference and non-reference insertions were analyzed using a COMPARE analysis  associatAlu insertions and associated gene names from RNA and protein COMPARE analysis were uploaded in batch to the MSigDb ‘Investigate Gene Sets’ from the Broad Institute Gene Set Enrichment Analysis web interface [-4. Interactome plots were used to visualize relationships between genes in pathways using Search Tool for the Retrieval of Interacting Genes/Protein (STRING) 9.0 [Gene loci containing candidate non-reference (polymorphic and singleton) LINE-1 and nterface  (using tING) 9.0 . Plots wp-value).To investigate preferential transposable element insertion in genes implicated in oncogenesis and mouse common insertion sites, we used a hypergeometric distribution test (pHypr R-package) which controlled for genes tiled on the array. Results were plotted using the –log(Tumor vs normal gene expression for genes containing candidate non-reference TE insertions was assessed for each tumor type using large tumor/normal gene expression databases. Tumor gene to normal gene expression ratios were obtained using NCBI GEO2R . GEO2R wExpression of STOP genes containing candidate non-reference TE insertions was assessed using log2 transformed Agilent mRNA expression data  obtained


KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression  were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5  and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.We investigated PD-L1 changes in response to MEK and AKT inhibitors in  Lung cancer is the leading cause of cancer death in developed countries with 1.8 million new lung cancer diagnoses occurring globally per annum  3] 4]  [4] 3] [  [4] 3] Programmed-death 1 and 2 (PD-1 and PD-2) are cell-surface receptors found on T-cells and their interaction with programmed-death ligand 1 and 2 (PD-L1 and PD-L2) on cancer cells provide an important inhibitory role in the immune response . PD-1 inKRAS mutations are found in 33% of advanced adeno-NSCLC [no-NSCLC . There ano-NSCLC  13]. De. DeKRAS no-NSCLC  15]. Dr. DrKRAS no-NSCLC .KRAS mutations . Th. ThKRAS gnalling . Others pression , 18, PD-2. Sensitivity of KRAS mutant cell lines to Trametinib (MEK inhibitor) or AZD5363 (AKT inhibitor)  including determination of GI50 was done using 72 hour sulforhodamine B assays. Cells were exposed to GI50 concentrations of trametinib and AZD5363 for 6hrs, 24hrs and 3 weeks.Cell lines were obtained from ATCC . Cell lines were cultured in RPMI supplemented with 20% fetal bovine serum and incubated in humidified atmosphere with 5% COFixation was achieved using methanol and 4% formalin followed by antigen retrieval ). A blocking step using bovine serum albumin (BSA), glycine and 0.02% triton for membrane permeabilisation, one hour, was performed. Anti-PD-L1 antibody , anti-rabbit  and anti-pan-cytokeratin  at 1:200 for 1 hour provided PD-L1 and cytokeratin labelling. Slides were mounted with Molecular Prolong, prolonged diamond antifade with DAPI  for nuclear identification. Analysis was using Bioview analysis-linked immunofluorescence microscope system . IN cell software  quantified fluorescent intensity of control and drugged samples.A jurkat cell line assay system (stably transfected with NFAT-luciferase reporter and human PD-1) was obtained from BPS Biosciences . Co-culture of the PD-1/NFAT reporter- jurkat cell line and a PD-L1 expressing cell line results in activation of NFAT-luciferase via TCR and MHC interaction. PD-L1 to PD-1 interaction downregulates the NFAT-luciferase. Thus the degree of decrease in luminescence is proportional to expression of PD-L1.Cell lines  were co-cultured with the jurkat cell line assay system  for 16 hours and the one-step luciferase assay system  added. Luminescence was measured on a luminescence plate reader . Results were produced in triplicate.Validation of the co-culture system was performed using a PD-L1 neutralising antibody  . Western blotting was performed to quantify MHC class I expression on cell lines .KRAS mutant cell lines . There were some temporal differences in the expression of PD-L1 at different time points, i.e. 6hrs, 24hrs and 3 weeks. However, we conducted experiments to evaluated functional aspects of PD-L1 expression only at 3 weeks as 3 weeks was considered to be a more relevant time point while exploring mechanisms of resistance to targeted small molecule drugs. All cell lines demonstrate PD-1/NFAT reporter- jurkat cell co-culture results in concordance with the immunofluorescence results with a decrease in luminescence corresponding to an increase in PD-L1 levels on immunofluorescence apart from H2291 on dosing with AZD5363 . Thus atEGFR mutant and ALK rearranged NSCLC cancers exposed to EGFR and ALK inhibitors. Azuma and coworkers demonstrated downregulation of PD-L1 by erlotinib when assessed by flow cytometry [EML4-ALK in Ba/F3 cells increased PD-L1 expression. PD-L1 expression in EML4-ALK-positive NSCLC cells was reduced by treatment with the ALK inhibitor alectinib and by RNA interference with ALK siRNAs. [KRAS mutations—studies demonstrate PD-L1 up regulation with KRAS mutations and potential mechanisms to be via AKT–mTOR signalling or ERK [Previous work has explored mechanisms of interaction between cellular pathways and immune pathways in ytometry . A studyytometry . Forced  siRNAs. . Data isg or ERK  18]..EGFR mutKRAS mutation, with targeted inhibition decreasing PD-L1 expression  PD- PD-23] Ppression  18]. Pr. PrKRAS pression . In thisKRAS mutant cell line panel studied, PD-L1 overexpression is not a consistently seen in response to MEK and AKT inhibitors.Our data revealed a small but functional increase and decrease in PD-L1 expression in 3/5 and 1/5 cell lines exposed to the MEK inhibitor trametinib respectively. Further when exposed to the AKT inhibitor AZD5363, functional increase and decrease in PD-L1 expression occurred in 2/5 and 2/5 cell lines respectively. Thus in the KRAS mutant cell lines relatively rapidly and economically. Functional validation of the jurkat co-culture system is provided by the high concordance seen between the immunofluorescence results and jurkat co-culture results. The functional nature of the jurkat co-culture system mitigate the inherent issues with immunofluorescence measurement alone. Immunofluorescence provides only a comparative measure of the uptake of antibodies by surface markers and makes no assessment of the functional interactions of PD-L1, which are the more significant elements when we consider potential biological function and resistance mechanisms. Immunohistochemistry is the most common method of measuring PD-L1 in the clinical context with well documented issues with standardization across platforms [Our approach of in-vitro exposure of cells to MEK and AKT inhibitors with functional read out of T cell activation allowed us to test PDL-1 expression as a putative mechanism of resistance in a significant number of latforms  and we platforms  26] and andKRAS In conclusion this work represent a novel interrogation of the late effects of signaling pathway inhibitors and immune pathway interaction. This shows small statistical changes in PD-L1 levels, which are not consistent across all cell lines studies.S1 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Targeting PRKCI by small molecule inhibitor, sodium aurothiomalate (ATM), significantly reduced the in vivo tumor growth and may be a new therapeutic strategy to improve the treatment of CCOC.Clear cell ovarian cancer (CCOC) is an epithelial ovarian cancer histotype with unique pathologic, biologic and clinical features. Despite its worse prognosis than serous ovarian cancer (SOC), the genomic landscape of CCOC is less well defined. Integrated genomic analysis of CCOC allows the identification of potential therapeutic targets to improve the treatment of this tumor. Using comparative genomic hybridization and gene expression profiling, we have screened 12 CCOC cell lines and 40 tumors to identify 45 amplified and overexpressed genes. Pathways analysis of these genes identified 19 genes with cancer-related functions. Of these, PRKCI is one of the most frequently amplified and overexpressed genes and its expression induced cancer cell proliferation and migration/invasion  Ovarian cancer is the most lethal of gynecologic cancers in the United States resulting in 14,000 deaths each year . The majClear cell ovarian cancer (CCOC) is the third most common subtype of ovarian cancer, with an estimated incidence of 5 % of all epithelial ovarian malignancies , 5. CCOCTP53), CCOC is characterized with various key functional mutations [ARID1A in approximately 50% of cases [PI3KCA and CTNNB1 conferring hyperactivated PI3K/Akt and Wnt/β-catenin pathway respectively [Recent studies have revealed distinct genomic landscape in CCOC. Unlike serous tumors which has relatively low mutation load  the identification of potential therapeutic target genes through an integrated genomics approach; and (2) a “proof-of-principle” demonstration that genes on the list impact ovarian clear cell cancer biology and can be potential therapeutic targets. Through integrated analyses of high-resolution array comparative genomic hybridization (aCGH) and microarray-based gene expression profiling data generated from CCOC cell lines and patient tumor specimens, we have generated a list of candidate genes with DNA copy number amplification associated with mRNA overexpression. The candidate genes were further screened for important cancer-related functions through bioinformatic annotation. This approach led to the identification of genes that were potential “drivers” for tumorigenesis of clear cell cancers of the ovary, a disease distinctive in clinicopathology and molecular biology to the high-grade serous carcinoma as the most common ovarian cancer subtype .The genomic DNA copy status of CCOC was investigated by high-resolution aCGH using Agilent human 105K oligonucleotide microarrays on 12 CCOC cell lines. Genomic copy number for each probe was determined by calculating the log2 ratio of median signal intensities of the cell lines and normal reference DNA. A genome-wide view of the copy number variation in the cell lines is shown in Figure p < 0.001. Among the 391 amplified genes, 45 of them were associated with mRNA overexpression , suggesting mRNA overexpression of PRKCI is due to amplification . Notably, the amplification and overexpression of PRKCI were found specifically in CCOC cell lines but not in SOC cell lines  and CCOC tumor specimens. Elevated PRKCI copy number was found in 20 out of 27 CCOC tumor specimens when compared with peripheral blood cell DNA as normal reference Figure . Conversp-value = 0.0309, log-rank test) .To delineate the potential prognostic impact of CCOC, an independent set of 40 CCOC tumor specimens  Figure . Consistin vitro cell growth  Figure , demonsth Figure , non-adhh Figure , cell mih Figure  and cellh Figure . In addis Figure . To furts Figure . PRKCI-oh Figure , non-adhh Figure , cell mih Figure  and invah Figure . In addih Figure  and 3M.in vitro and investigate its potential use in vivo. Administration of 25 µM ATM significantly suppressed adherent cell growth on CCOC cells with high PRKCI level, including KOC-7c and OVISE control cells and PRKCI-overexpressing ES2 and TOV21G cells , was utilized to validate the biological functions of PRKCI s Figure  and 4E. s Figure  and 4H. s Figure  and 4F.in vivo tumor growth, PRKCI-knockdown and PRKCI-overexpressing CCOC cells were intraperitoneally injected into athymic nude mice and the mice were administrated with 20 mg/kg/day for 14 consecutive days. ATM treatment was found to suppress tumor growth in mice with PRKCI overexpressing cells, including mice with KOC-7c control cells  in CCOC. The gene list was filtered using PathwayStudio software to identify genes with cancer-related biological function. The 19 genes identified are enriched for potential ‘driver” genes whose function is important for the biology and clinical features of the tumor. In addition, “driver” genes can be potential therapeutic targets. This approach of an integrated genomic analysis has been shown to be effective at identifying novel targets in other tumors -19.versus normal uterine endometrium, significant upregulation was observed for PRKCI in ovarian endometriosis  [p = 0.0021, Our integrated genomic approach has clear limitations in that it could underestimate the number of “driver’ genes in CCOCs. Recently, a comprehensive study interrogating the robustness of 12 aCGH platforms has revealed aCGH may miss 20-70% of CNV events when manufacturer recommended bioinformatics workflow is conducted (as performed in this study) . Unfortu 0.0001) . When coPIK3CA, SOX2 and ECT2. Amplification of 3q26 is one of the most prominent copy number variation features that potentially drives the tumorigenesis in several types of solid tumors [in vivo growth. Moreover, the clinical relevance of PRKCI in CCOC is further implicated by its specific small molecule inhibitor sodium aurothiomalate (ATM), which efficiently abrogates the oncogenic effects of PRKCI in CCOC cell lines in vitro and in vivo.As proof of principle, we analyzed in detail the function of PRKCI in CCOC progression, which is one of the most frequently amplified and highly overexpressed genes. Until recently, the biological significance of PRKCI in clear cell ovarian carcinoma remains largely unknown. Nevertheless, integrated genomic and phenotypical analyses of PRKCI in CCOC clinical specimens have suggested PRKCI has the hallmarks of a driver gene in CCOC Figure -4. PRKCId tumors , includid tumors . Howeverd tumors . Rather,d tumors . ConversPI3KCA or deletion of PTEN, both of which lead to excessive generation of PIP3 to facilitate PRKCI mediated biology. It is thus hypothesized that a CCOC clone with PRKCI amplification and overexpression may be more favored in clonogenic selection for its tumorigenic predisposition.The biological significance of PRKCI in CCOC is further suggested by the distinctive molecular context of this ovarian cancer subtype. PRKCI represents a structurally and functionally distinct subclass in the protein kinase C family which is less responsive to receptor tyrosine kinase or G-protein coupled receptor initiated diacylglycerol or calcium signals. One of the major second massagers activating PRKCI is phosphatidylinositol 3,4,5-trisphosphate (PIP3), the downstream product of PI3 kinase , throughThe PRKCI inhibitor ATM binds to the Cys-69 of PRKCI. Cys-69 a unique amino acid residue found in the PB1 domain of PRKCI and binding of ATM to Cys-69 of PRKCI inhibits PRKCI from interacting with Par-6 and p62. ATM specifically inhibits protein-protein interaction of PRKCI but not other PB1 domain containing proteins, including p62-p62, p62-NBR1 and MEKK3-MEK5 interactions . ATM canThis study is the first report of an integrated genomic analysis of CCOC through which we have identified for the first time PRKCI as a potential target gene in human clear cell ovarian cancer. PRKCI was copy number amplified and mRNA overexpressed in CCOC cell lines and patient specimens. Higher PRKCI levels were associated with increase in cell proliferation, metastatic potential and tumorigenicity of CCOC cells. These findings also support the potential use of PRKCI inhibitor, ATM, as a therapeutic agent to improve the treatment of human clear cell ovarian cancer.The human CCOC cell lines KOC-7c, OVISE, OVSAYO, ES-2, TOV21G, RMG1, KK, OVMANA, SMOV2, HAC2, OVTOKO and OVAS were maintained in RPMI 1640 medium supplemented  with 10% FBS, 1% L-Glutamine and 1% penicillin/streptomycin . Immortalized ovarian surface epithelial (OSE) cell lines, IOSE-80 and IOSE-120, were maintained in a 1:1 mixture of medium 199  and medium 105 , supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine. ES-2, TOV21G and RMG1 cell lines were purchased from American Type Culture Collection. KOC-7c, OVISE, OVSAYO, KK, OVMANA, SMOV2, HAC2, OVTOKO and OVAS were kindly provided by Professor Hiroaki Itamochi at Tottori University School of Medicine, Japan.in vitro assays previously described [The establishment of stable cell lines was reported in previous publication . Brieflyescribed .https://clinicaltrials.gov/ct2/show/NCT00897442?term=GOG+0136&rank=1). 10 microdissected CCOC specimens and 10 normal ovarian surface epithelium (OSE) cytobrushing specimens for RNA extraction and microarray analysis were obtained from primary tumors of untreated ovarian cancer patients and healthy donors respectively at the Brigham and Women’s Hospital . 40 FFPE tumor specimens from patients with clear cell ovarian cancer for immunohistochemical analysis of PRKCI and Kaplan-Meier analysis of patient survival according to PRKCI expression level were obtained from Tottori University School of Medicine .27 paired normal (Buffy coat genomic DNA obtained from the same patient) and clear cell ovarian tumor genomic DNA specimen for copy number variation analysis were obtained from GOG-0136. Detailed information of GOG-0136 is available at the NCI webpage . PRKCI copy number variation qPCR was performed with iQ SYBR Green Supermix  according to manufacturer’s protocol using the following primers: Forward: 5’-GAGAGACTCGCCTCCTGAG-3’ and Reverse: 5’-ACTCGCAACCCCAAATACACA-3’. DNA content was normalized to that of Line-1, a repetitive element for which copy numbers are similar between normal and neoplastic cells. Relative DNA copy number was determined by normalizing to that of pooled normal gDNA sample. PRKCI mRNA expression qRT-PCR was performed with the One-Step qRT-PCR with SYBR Green kit  using the primers: Forward: 5’-TACGGCCAGGAGATACAACC-3’ and Reverse: 5’-AAGAGCCCACCAGTCAACAC-3’. mRNA content was normalized to that of the housekeeping genes GAPDH, GUSB, and cyclophillin. Relative mRNA expression was determined by normalizing to that of the average of the two immortalized OSE cell lines. Relative expression of PRKCI was determined by comparing the expression levels of CCOC lines with those in the 2 normal OSE cell lines.Immunochemistry (IHC) on FFPE xenografts and patient tumor specimen were performed using primary antibodies against human PRKCI , human phospho-ERK1/2  and human Ki-67  as described .PRKCI protein expression grading was conducted by determining the percentage and intensity of positive cells in 3 different areas at low-power magnification. First, the percentage of positive cells in each section was scored with a 5-point scale: 0 for < 5%, 0.25 for 5%-25%, 0.5 for 26%-50%, 0.75 for 50%-75%, and 1 for over 75%. Second, the intensity of positive signal was scored with a 3-point scale: 1 for weak staining, 2 for moderate staining, and 3 for intense staining. The weighed score of PRKCI staining intensity of each section was obtained by multiplying the percentage score by the intensity score (the maximum weighed score is 4). The IHC quantification was performed by 2 individuals, including 1 pathologist for independent IHC scoring. The average score was used for downstream analyses. The scoring was done blinded to the clinical data.6 ES2 cells, 4 x 106 TOV21G cells or 5 x 106 KOC-7c cells were injected). Mice were sacrificed 3-6 weeks after tumor cell injection. Sodium aurothiomalate (ATM) dissolved in 1X PBS was administrated intramuscularly at the thigh of the mice starting from 1-3 week after injection of CCOC cells at a dose of 20 mg/kg/day for 14 consecutive days. Equal volume of 1X PBS solution was employed as control. Mice were sacrificed after treatment. In all in vivo experiments, tumor burden was assessed by the total tumor weight at the time of sacrifice.Female 4-6 weeks old athymic nude mice were purchased from Charles River Laboratories. CCOC cells were intraperitoneally injected into mice (For each mouse, 1 x 10


The relative length of the second and fourth fingers (2D:4D) may be a sex-linked correlate of prenatal androgen exposure. However, the nature of the sex-linkage is controversial, with evidence for both X- and Y-linkage of the 2D:4D phenotype.To examine transgenerational effects relating to sex-linkage. In addition, assortative mating on 2D:4D was considered, as well as associations between 2D:4D and age and sex.A family study was conducted. Parents and offspring completed a demographic questionnaire, and digit ratios were calculated from photocopies of participants' hands.We recruited and phenotyped 585 individuals attending a cultural festival in Wales. 2D:4D information was available for 47 mother-son dyads, 70 mother-daughter dyads, 31 father-son dyads and 30 father-daughter dyads.Correlations between 2D:4D of parents and children, as well as between mothers and fathers were conducted. 2D:4D was also examined in relation to age and sex.There was a sex difference in 2D:4D . Within the dyads, there was a significant positive correlation between mother and daughter 2D:4D, but no significant correlation between mother and son ratios, nor between father and offspring ratios. The overall pattern of correlations (with emphasis on father-son dyads) was not supportive of Y-linkage. There was a positive correlation between 2D:4D and age in children, and a negative correlation between 2D:4D and age in adults, and no evidence of assortative mating. Our data are consistent with the notion of 2D:4D as a sexually-dimorphic, mildly age-sensitive, and transgenerationally-transmitted trait that is more likely to be X- than Y-linked. •Digit ratio (2D:4D) may be a sex-linked correlate of prenatal sex hormone exposure.•2D:4D was examined in a family design study of a general population sample.•Correlations for 2D:4D within parent-child dyads were more consistent with X- rather than Y-linkage.•There were no significant correlations between 2D:4D of mothers and fathers.•2D:4D correlated positively with age in children, and negatively with age in adults. Correlations across male family members (r = 0.305–0.543) appeared to be stronger than those across female family members (r = 0.217–0.299), indicative of a Y-linked genetic effect.Twin studies have indicated that the 2D:4D ratio may be partially influenced by genetic factors. For instance, Hiraishi et al. r = 0.28–0.35) they did not find a significant association between maternal and newborn son 2D:4D (r = − 0.03-0.227); statistical comparison of these two correlations suggests no significant difference. Likewise, although Ramesh and Murty Comparing the findings of Phelps r = 0.18–0.19) within couples, a result consistent with the notion that individuals who experience similar prenatal environments may be attracted to one another. However, although Manning β = .15, p = .03), Hauth et al. In addition to being partially heritable, the 2D:4D ratio may represent a surrogate marker of assortative mating: Voracek, Dressler, and Manning In the present exploratory study, we collected 2D:4D data on a moderately-sized sample of participants (parents and children) from the general population recruited at a Welsh cultural festival. The study had four main aims: i) to confirm the well-established sex difference for the 2D:4D ratio, ii) to examine possible relationships between digit ratio and age, iii) to test for relationships between 2D:4D in mothers and their male and female offspring, and fathers and their male and female offspring, and iv) to test for relationships between 2D:4D ratios in partners with one or more shared children.22.1Members of the general population were recruited and tested at a stand held by the British Psychological Society's (BPS) Welsh Branch at the National Eisteddfod in Abergavenny, South Wales, between July 30th and August 6th 2016. Participants volunteered to take part in the study and were not provided with any monetary or other incentive.2.2The current research was reviewed by Cardiff University School of Psychology Ethics Committee , and was conducted in accordance with the Declaration of Helsinki. After reading an Information Sheet, adult participants provided written consent to continue with the study, whilst written parental assent (as well as child consent) was obtained for minors to participate. A brief demographic questionnaire was then used to obtain information on participants' sex, age, ethnicity, and relationship to anyone else participating in the study. A Hewlett Packard ENVY 4500 portable photocopier was used to scan participants' left and right hands, and greyscale images  were made at a resolution of 300 ppi. These images were saved, and 2D:4D ratios were subsequently calculated for each hand using AutoMetric 2.2 for Windows 2.3ICC = 0.929, F = 28.47, p < 0.001, and L2D:4D, ICC = 0.917, F = 23.192, p < 0.001. Mean R2D:4D and L2D:4D values were computed from these two sets of measurements, and were used in all subsequent analyses.2D:4D ratios were calculated twice , and intra-class correlation coefficients  were conducted to analyse reliability of measurement. Repeatability was determined to be high for both R2D:4D, U test as appropriate, and correlations were analysed using Spearman's test. For ease of comparison with findings from other studies, and because they are relatively robust to deviations from normality, correlation coefficients obtained by Pearson's test are also reported. Although very few notable differences are observed between the parametric and non-parametric analyses, interpretations of the findings are based upon non-parametric tests unless otherwise stated. The strength of correlations is compared statistically using Fisher's r-to-z transformation. p-Values < 0.05 were considered as being nominally significant; as the study was largely exploratory in nature, no corrections were made to take into account multiple testing issues. Data are presented as mean values (M) with standard deviation (SD) of the mean.As 2D:4D ratios were not normally-distributed (as indexed by Shapiro-Wilk test), between-group comparisons were analysed using one or two-tailed Mann-Whitney 33.1M = 40.14, SD = 22.77) completed the study. 2D:4D ratio information was obtained for 47 mother-son dyads, 70 mother-daughter dyads, 31 father-son dyads and 30 father-daughter dyads; information was available for 17 father-mother-son triads, and for 17 father-mother-daughter triads.585 participants  of predominantly White European ethnicity (97.4%) and aged 5-89 yrs (3.2rho (578) = 0.636, p < 0.001 , as well as within males, rho (234) = 0.626, p < 0.001  and females separately, rho (331) = 0.617, p < 0.001 . According to generally accepted criteria, the magnitude of these correlations was strong .There was a highly significant positive correlation between R2D:4D and L2D:4D across the total sample, 3.3M = 0.955, SD = 0.033; females, M = 0.968, SD = 0.031, U = 30,249.5, p < 0.001) and L2D:4D . Fathers (n = 50) had significantly lower R2D:4D ratios than mothers (n = 106) , and the same pattern of results was obtained for L2D:4D . Male offspring (n = 61) had significantly lower R2D:4D ratios than female offspring (n = 83) , whilst a similar trend for L2D:4D was not significant . R2D:4D was significantly higher than L2D:4D in females , although no such effect was observed in males .Across the overall sample, as expected, there were highly significant effects of sex on R2D:4D , but also separately for adults (≥ 18 yrs) and children (≤ 17 yrs). Although the effects were not statistically significant in the overall sample, we noted an interesting dissociation by which significant positive correlations between age and 2D:4D ratio were observed for the right hand of boys and the left hand of girls, and significant negative correlations between age and 2D:4D ratio were observed in adult males  see . The obs3.5We found evidence for highly significant associations between maternal 2D:4D ratios and 2D:4D ratios across all offspring; when offspring were sub-divided by sex, it was clear that the initial observed association was primarily being driven by a positive correlation between maternal and daughter 2D:4D ratios  > Mother-Daughter > Father-Son, with the last of these relationships most likely being negligible or nil If X-linkage is present, we would expect the following differences in rho = 0.276) and Mother-Son (rho = 0.099), Z = 0.74, p = 0.459. The slope for Mother-Daughter (rho = 0.283) was not significantly different from either the slope for Father-Daughter (rho = 0.276), Z = 0.03, p = 0.976, or Mother-Son (rho = 0.099), Z = 0.99, p = 0.322. However, the slopes were significantly different for Mother-Daughter (rho = 0.283) and Father-Son (rho = − 0.231), Z = 2.34, p = 0.019. As the former showed a positive correlation and the latter a negative correlation, no evidence was found for Y-linkage.For R2D:4D, there was no significant difference between the slopes for Father-Daughter (rho = − 0.171) and Mother-Son (rho = 0.151), Z = − 1.33, p = 0.184. However, although the Mother-Daughter (rho = 0.351) correlation did not differ from that of Mother-Son (rho = 0.151), Z = 1.11, p = 0.267, it was significantly different from Father-Daughter (rho = − 0.171), Z = 2.37, p = 0.018, the former being positive and the latter being negative. However, the slope for Mother-Daughter (rho = 0.351) did not differ significantly from that of Father-Son (rho = 0.058), Z = 1.37, p = 0.171.For L2D:4D, there was no difference between slopes for Father-Daughter (r = 0.276) and Mother-Son (r = 0.328) did not differ, Z = − 0.68, p = 0.497. Mother-Daughter (r = 0.293) did not differ significantly from Father-Daughter (r = 0.276), Z = 0.23, p = 0.818, or Mother-Son (r = 0.328), Z = − 0.49, p = 0.624. Mother-Daughter (r = 0.293) also did not differ from Father-Son (r = 0.363), Z = − 0.91, p = 0.363. For L2D:4D, Father-Daughter (r = 0.131) did not differ from Mother-Son (r = 0.194), Z = − 0.77, p = 0.441. Although Mother-Daughter (r = 0.299) was no different from Mother-Son (r = 0.194), Z = 1.41, p = 0.159, it was actually significantly stronger (positive) compared with Father-Daughter (r = 0.131), Z = 2.19, p = 0.029. Importantly, as with the analysis for R2D:4D, Mother-Daughter (r = 0.299) was not different from Father-Son (r = 0.305), Z = − 0.08, p = 0.936, so no strong evidence was provided for digit ratio being a Y-linked trait.Although Voracek and Dressler r rather than rho values from our data are used). There was a significant difference for Father-Son correlations for R2D:4D between the current study (r = − 0.193) and Voracek and Dressler (r = 0.363), Z = − 2.87, p = 0.004, with the former observing a non-significant negative correlation, whist the latter observed a significant positive correlation. For L2D:4D the slope observed in the current study (r = 0.119) was not significantly different from that observed by Voracek and Dressler (r = 0.305), Z = − 0.97, p = 0.332.We tested to see whether the Father-Son correlations differed between the data presented in the current study and those of Voracek and Dressler 3.6rho (21) = − 0.207, p = .343 ; L2D:4D, rho (21) = − 0.171, p = .436 . Furthermore, R2D:4D for mothers did not correlate with L2D:4D for fathers, rho (21) = − 0.083, p = 0.707 , and R2D:4D for fathers did not correlate with L2D:4D for mothers, rho (21) = − 0.029, p = 0.897 .We did not identify any significant correlations between digit ratio of spouses for either hand: R2D:4D, 4Previous research has indicated that the 2D:4D ratio, an index of prenatal and/or early postnatal sex hormone exposure, differs significantly between sexes and by age, is correlated in parent-offspring dyads (notably mother-daughter), and may be a marker of assortative mating. Here, we attempted to examine these initial findings in a moderately-sized exploratory family-based study with participants recruited from the general population of Wales.4.1We observed lower ratios in males than females for adults and younger participants, although the effect relating to L2D:4D in offspring was not statistically significant. The direction, and magnitude, of these findings is consistent with an extensive and robust previous literature 4.2We found preliminary evidence for associations between 2D:4D and age, although these should be viewed cautiously in light of the absence of correction for multiple testing and the fact that digit ratio measurements were taken from scanned images and therefore may not completely recapitulate real-life measurements due to induced minor tissue distortion 4.3Both left and right-hand maternal 2D:4D ratios correlated positively with left and right-hand 2D:4D ratios of their daughters (but not their sons), an observation which parallels the findings of Ventura et al. We found no evidence that 2D:4D ratios were correlated (either positively or negatively) between mothers and fathers with at least one shared child. This result is consistent with some previous research Whilst our participant sample is of a reasonable size, and is somewhat representative of the general UK population, the present findings should be treated with an appropriate degree of caution. As many participants were unrelated to each other, and because considerably more mothers than fathers took part, some of our analyses are underpowered. Moreover, our sample may be biased by ascertainment being selected from festival attendees with an interest in science. There is some evidence to suggest that individuals (and family members) attracted to scientific pursuits may exhibit relatively high levels of autism-related traits In conclusion, our data provide further evidence that the 2D:4D ratio differs, on average, between sexes and throughout development, and that it is substantially influenced by trans-generational factors. The effects observed were more consistent with X- rather than Y-linkage. Although we did not demonstrate evidence for 2D:4D ratios being correlated between partners with a shared child (a possible index of assortative mating), inconsistent findings in the literature suggest that the topic warrants further investigation.European Human Behaviour and Evolution Association (EHBEA), which was awarded to GR. The work was partially undertaken within the Medical Research Council UK Centre for Neuropsychiatric Genetics and Genomics (MR/L010305/1). The funders played no role in study design, data collection, analysis and interpretation, writing of the manuscript, or the decision to submit the article for publication.This work was supported by a Student Research Grant from the None.GR wrote the research proposal, designed the study, wrote the statistical analysis plan, collected and analysed the data, and drafted the initial manuscript. WD contributed to statistical analysis and interpretation, and WB translated English language questionnaires into Welsh. Both WD and WB revised the paper for important intellectual content. All authors read and approved the final article prior to submission.


Collectively, these cell lines show all the hallmarks of highly aggressive, metastatic breast cancer and are being used to evaluate combination therapy with alpha-particle emitter labeled HER2/neu reactive antibodies.Preclinical evaluation of therapeutic agents against metastatic breast cancer require cell lines and animal models that recapitulate clinical metastatic breast cancer as much as possible. We have previously used cell lines derived from the neu-N transgenic model to investigate anti-neu targeting of metastatic breast cancer using an alpha-emitter labeled antibody reactive with the rat variant of HER2/neu expressed by the neu-N model. To investigate alpha-particle emitter targeting of metastatic breast cancer using clinically relevant, commercially available anti-HER2/neu antibodies, we have developed cell lines derived from primary tumors and lung metastases from HuHER2 transgenic mice. We extracted primary mammary gland tumors, isolated the epithelial breast cancer cells, and established seven different cell lines. We also established 2 different cell lines from spontaneous lung metastases and cell lines from a serial transplantation of tumor tissues in HuHER2 transgenic mice. HuHER2 protein was overexpressed in all of the established cell lines. The mRNA level of ER (estrogen receptor) and PR (progesterone receptor) was relatively low in the cell lines compared to normal mammary gland (MG). As EMT markers, the expression of E-Cadherin in the cell lines was downregulated while the expression of TWIST1 and Vimentin were upregulated, relative to MG. Furthermore, trastuzumab directly inhibited cellular viability. Biodistribution studies with  Trastuzumab (Herceptin), an anti-human HER-2 IgG monoclonal antibody was obtained from Genentech .All chemicals were purchased from Sigma-Aldrich Chemical Co.  or Thermo Fisher Scientific , unless otherwise specified. Aqueous solutions were prepared using ultrapure water  treated with Chelex resin purchased from Bio-Rad Laboratories, Inc. . p-SCN-Bn-DTPA was purchased from Macrocyclics, Inc. . Indium-111  was added to the minced tissue. The tissue was digested by rotating in a Hybaid oven at 37°C for at least one hour. When digestion was completed the digestive enzymes were inactivated by additional media. The mixture was centrifuged at 1500 rpm for 5 minutes at 4°C. Red blood cells were removed by serial resuspension and centrifugation using lysis buffer. Fibroblast cells were removed by brief trypsin incubation during several passages.SKBR3 and BT474 cell lines were obtained from the American Type Culture Collection . pGL4.50[luc2/CMV/Hygro] vector was purchased from Promega  and transfected into the cell line using Lipofectamin 2000 as describe in the manufacture’s protocol. Luciferase reporter stable cell line was selected by 25 μg/ml of Hygromycin B (Thermo fisher).Mice (2-6 months) were bred as both a monogamous pair  and a polygamous trio  breeding scheme. Offspring were weaned at 21 days. On the day of weaning, a small piece of tail (5mm or less) was snipped with sterile scissors and the DNA was extracted using STE lysis buffer. 0.5 μl of DNA was used for genotyping via PCR. A primer set specific to human growth hormone exons 4 and 5 (forward -5’-CCTCCTAAAGGACCTAGAGGAAGGC-3’ and reverse -5’-CAAGGCCAGGAGAGGCACTGGGGAG-3’) was used -trans-cyclohexane-1,2-diamine-N,N′,N′,N″,N″-pentaacetic acid (p-SCN-Bn-DTPA) and purified by size exclusion. The resulting antibody conjugate was added to an acid washed 1.5 mL Eppendorf tube containing [111In]InCl3 (37-74 MBq), 0.25 mL of 0.2 M HCl and 0.03 mL of 3 M NH4OAc, pH=7. The resulting mixture was allowed to set at room temperature for 45-60 minutes then purified as described previously. Radiochemical purity was determined by thin layer chromatography (TLC), and the protein concentration was determined by Nanodrop .The radiolabeled anti-HER2 antibody was prepared as previously described . Briefly


By whole exome sequencing of the lines and matched patient blood samples, we demonstrate that the mutational spectrum of the lines is representative of primary OSCC in The Cancer Genome Atlas. We show that loss of function mutations in FAT1  and CASP8 (Caspase 8) frequently occur in the same tumour. OSCC cells with inactivating FAT1 mutations exhibited reduced intercellular adhesion. Knockdown of FAT1 and CASP8 individually or in combination in OSCC cells led to increased cell migration and clonal growth, resistance to Staurosporine-induced apoptosis and, in some cases, increased terminal differentiation. The OSCC lines thus represent a valuable resource for elucidating the impact of different mutations on tumour behaviour.Oral squamous cell carcinoma (OSCC) is genetically highly heterogeneous, which contributes to the challenges of treatment. To create an  •Generation of human oral squamous cell carcinoma lines with whole exome sequence data.•Mutational spectrum of the lines is representative of primary tumours.•Loss of function mutations in FAT1 and CASP8 frequently co-occur.•Inactivating FAT1 mutations correlate with reduced intercellular adhesion.•Inactivation of FAT1 and CASP8 results in increased clonal growth, migration and apoptosis resistance. The global annual incidence of head and neck squamous cell carcinoma (HNSCC) is approximately 600,000, with over 300,000 deaths attributable to the disease each year Previous studies have used next-generation whole exome sequencing of primary tumours to reveal the mutational landscapes of human HNSCCs and suggest novel oncogenic drivers to target for potential therapies Oral cancer is a subtype of HNSCC, comprising tumours of the lip and oral cavity. Worldwide, over 250,000 new cases and 125,000 deaths from oral cancer occurred in 2008 As in other solid tumours NOTCH1, CASP8 (Caspase 8) and FAT1  FAT1 mutation and overall survival in HNSCC differs according to the HPV status of the tumour Cells can readily be cultured from human oral tumours and normal oral mucosa, and several well-annotated collections of HNSCC cells are available CASP8 and FAT1 on cell behaviour.In this study, we set out to develop new OSCC lines, discover which mutations are tumour-acquired and determine whether they are indeed representative of mutational burden in primary tumours. We then used the lines to explore the impact of mutations in Anonymized biopsies of OSCC or normal oral mucosa were collected with appropriate ethical approval . Cells were isolated and cultured on a feeder layer of J2 3T3 cells in complete FAD medium as described previously Genomic DNA was extracted from OSCC lines (passage 2–4) and patient-matched blood. Whole exome sequencing was performed by Beijing Genomics Institute (Hong Kong). Raw image files were processed by Illumina base calling Software 1.7 or base calling with default parameters, and the sequences of each individual were generated as 90 bp paired-end reads. High-quality reads were aligned against the NCBI human reference genome (hg19) using Burrows-Wheeler Aligner (v0.5.9) with default parameters. Picard (v1.54) was employed to mark duplicates and was followed by Genome Analysis Toolkit  to improve alignment accuracy. Putative somatic single nucleotide variations (SNVs) were predicted by VarScan2.25 with the parameters as “-- min-coverage 5 --min-coverage-normal 5 --min-coverage-tumour 5 --min-var-freq 0.1 --min-freq-for-hom 0.75 --min-avg-qual 0 —somatic-p-value 0.15”. In order to obtain high confidence somatic SNVs, an in-house pipeline was applied. Somatic InDels were predicted by GATK SomaticInDelDetector with default parameters. A pipeline was developed to obtain high confidence somatic InDels; normal and tumour bam were reused to perform local realignment and germline indels were filtered for high confidence indels, with normal coverage and tumour coverage no less than 5. High confidence somatic single nucleotide variants and InDels were annotated using ANNOVAR. Functional impacts of missense mutations were predicted using SIFT, PolyPhen2, PhyloP, MutationTaster and LRT annotations.The Oncodrive-fm method was applied, as previously published, to identify significantly mutant genes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways cBioPortal.org. KEGG pathway analysis was performed; –log2(P) values were calculated and presented in heat maps created using GENE-E software (Broad Institute). OncoPrint, mutation diagrams and tests for mutual exclusivity of genomic alterations were downloaded from cBioPortal.Whole exome sequencing data from The Cancer Gene Atlas (TCGA) HNSCC collection RNA was harvested using the RNeasy Mini Kit and QIAshredder columns according to the manufacturer's instructions (Qiagen). cDNA was synthesized using SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen). qPCR was performed using TaqMan Fast Universal PCR Master Mix without AmpErase UNG (Life Technologies) on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with the following primers: CASP8 (Hs01018151_m1), FAT1 (Hs00170627_m1) and GAPDH (Hs02758991_g1) as the internal control (Life Technologies). Relative quantification was performed using the 1/ΔCt or ΔΔCt methods.® Negative Control No. 1 (Thermo Fisher) siRNAs were used. Prior to transfection, wells were coated with 20 μg/mL Rat Tail Collagen I (Corning) and incubated for 1 h at 37 °C. Cells were transfected using either Lipofectamine RNAiMax or Lipofectamine 2000 with Opti-MEM medium (Thermo Fisher). The viability of transfected cells was confirmed by Trypan Blue exclusion prior to performing cell behaviour assays.SMARTpool ON-TARGETplus CASP8 and FAT1  and Silencer48 h after transfection, cells were seeded at clonal density in 6-well plates on a layer of feeders. 10–14 days after seeding, feeders were detached and keratinocytes were fixed in 10% formalin and stained with a solution of 1% Rhodamine B and 1% Nile blue. Well images were acquired on the Gel Doc™ XR+ (Bio-Rad). Percent well area covered by cells was determined using the ColonyArea plugin ® ZOOM (Essen BioScience). Area closure was quantified in ImageJ, following the manufacturer's instructions.24 h after transfection, cells were seeded on collagen-coated well plates in the Oris™ Cell Migration Assay (Platypus Technologies) and cultured in complete FAD medium. 24 h after plating, cell seeding stoppers were removed. Phase-contrast images were acquired for 16–24 h using an IncuCyte24 h after transfection, cells were seeded on collagen-coated 96-well plates in complete FAD medium. 24 h later cells were incubated with varying concentrations of Staurosporine  in complete FAD medium for 4 h at 37 °C.® 488 conjugated anti-cleaved Caspase-3 (Asp175) antibody  or mouse anti-transglutaminase 1 . Cells were incubated for 1 h at room temperature in blocking buffer with the following secondary antibodies, as appropriate: Alexa Fluor® 488 goat anti-rabbit IgG H + L  or Alexa Fluor® 555 goat anti-mouse IgG H + L . Plasma membranes and nuclei were counterstained with CellMask™ Deep Red  and DAPI (1 μg/mL in PBS), respectively, for 1 h at room temperature. Following Caspase-3 labelling cell nuclei were counterstained with DRAQ5™ . Images were acquired using the Operetta® High-Content Imaging System (PerkinElmer). Positive nuclear Ki67 and cytoplasmic TGM1 immunostaining were quantified using an in-house pipeline on Harmony® software (PerkinElmer), while Caspase-3 positive cells were quantified using the “Apoptosis-1” module (PerkinElmer).Cells were fixed in 4% paraformaldehyde, permeabilized using 0.5% Triton X-100 (Sigma-Aldrich) in PBS and blocked in a solution of 10% fetal bovine serum (Thermo Fisher), 0.25% gelatin from cold water fish skin (Sigma-Aldrich) and 0.5% Triton X-100 in PBS. Cells were incubated overnight at 4 °C with the following primary antibodies in blocking buffer: rabbit anti-Ki67 , Alexa FluorP value of <0.05 was considered significant, unless otherwise noted.Data were analyzed using an unpaired Student's t-test or one-way ANOVA with Tukey's Multiple Comparison Post Test on GraphPad Prism 5.0b, and a We derived multiple low passage polyclonal cell lines from primary oral squamous cell carcinoma biopsies by culture on a 3T3 J2 feeder layer in order to minimise selection for rapidly dividing cells Ten of the 16 cell lines were derived from cancers of the tongue, four from the alveolus, one from the buccal mucosa and one from the hard palate . No assoQ values < 0.10. Only two genes were found to be significantly mutated – the canonical tumour suppressor TP53  and CSMD2 . While CSMD2 has a tumour suppressive role in several cancers CSMD2 mutations in HNSCC have not been described previously.To identify putative driver genes and pathways that, when mutated, contribute to OSCC tumourigenesis, the Oncogene-fm method was applied P value < 0.10 (FAK) is a key mediator of extracellular matrix interactions and cellular signalling, contributing to HNSCC onset and progression Twenty KEGG pathways were significantly mutated across the 16 OSCC lines . Unsurpre < 0.10 . Common NOTCH1 mutations (found in 10–15% of HNSCC) NOTCH1 mutations were identified in three of our OSCC lines via whole exome sequencing  mutations  Network published genomic profiles for 279 HNSCCs quencing D. In addutations . 44% of To discover whether the mutational spectrum of OSCC is distinct from that of other HNSCCs, we determined the site of origin of each of 267 tumours in TCGA and then performed unsupervised clustering of the samples according to the significance of mutated KEGG pathways E. This iTo determine whether the KEGG pathways that were mutated in the OSCC lines were similar to those in primary tumours, we performed unsupervised clustering of TCGA OSCC samples ranked according to the frequency of mutated KEGG pathways in the cell lines. As shown in PLEC, CASP8, SYNE2, FAT1, LRP1B, COL11A1 and HUWE1 , and SYNE2 and LRP1B  were identified as frequently co-mutated in HNSCC.To evaluate the impact of mutations and expression of putative driver genes established by genomic studies on OSCC cell behaviour, we conducted a systematic literature review of genes implicated in homoeostasis or disease in stratified squamous epithelia through regulation of adhesion, invasion or motility. We focused on genes that were members of multiple mutated non-tissue specific KEGG pathways and/or were most significantly mutated in our study and TCGA. We prioritized those genes harbouring mutations in more than 8% of HNSCCs and at least two of our cell lines. Seven genes were identified – nd HUWE1 A. We alsnd HUWE1 B. CASP8 n = 33), hypopharynx (n = 2) and larynx (n = 72). Considering copy number alterations and truncating, missense and in-frame mutations, 23% of tumours of the oral cavity harboured mutations in FAT1 alone, 5.8% in CASP8 alone and 9.3% in both FAT1 and CASP8, compared with 22%, 2.8% and 1.9%, respectively, in tumours of other areas of the head and neck. The increased frequency of CASP8 and FAT1 co-mutations in OSCC relative to other HNSCC, recent evidence suggesting functions of CASP8 that extend beyond programmed cell death FAT1 in oncogenesis Of the 279 patients with HNSCC reported by TCGA in 2015 FAT1 across four of the 16 OSCC lines, and three in CASP8 across three lines . In addition, FAT1 mRNA levels were higher in normal oral keratinocytes than OSCC lines , whereas in others knockdown of CASP8 and FAT1 individually or in combination was equally effective .Given the association between adhesion A and betinocytes F. In somCASP8 and FAT1 knockdown on clonal growth could not be accounted for by an increase in proliferation, and in several cases Ki67 labelling was, rather, reduced (CASP8 and FAT1 did not have a consistent effect on the proportion of cells that underwent terminal differentiation, as judged by the percentage of cells expressing Transglutaminase 1: there was no effect of double knockdown on SJG-17 cells; differentiation was stimulated in SJG-33 cells; and decreased in SJG-41 cells (CASP8 and FAT1 did, however, increase resistance to Staurosporine-induced apoptosis (The impact of  reduced A. Knockd41 cells B. Knockdpoptosis , as judgpoptosis C.CASP8 and FAT1 are likely to confer a growth advantage on OSCC by reducing cell–cell adhesion, promoting cell migration and conferring resistance to apoptosis. Furthermore, as expected, knockdown tended to have more pronounced effects in cells with wild type CASP8 and FAT1.We conclude that loss of function mutations in NOTCH1-3in vitro and in xenograft assays.While many investigators have generated cell lines from human HNSCC and used them to study the biology of this tumour type CASP8 and FAT1, two genetic changes that, we found, frequently occur together in individual OSCC. We found that in the lines, as in primary tumours, inactivating mutations in CASP8 did not result in a reduction in CASP8 expression, whereas FAT1 mutation did correlate with reduced mRNA levels. While FAT1 mutant lines showed reduced intercellular adhesion, FAT1 knockdown in a wild type line was not sufficient to cause a significant change in morphology. It will therefore be of interest to discover whether the effect of FAT1 loss is dependent on other OSCC mutations (PTEN will affect the actin cytoskeleton AJUBA mutations are likely to impact E-cadherin mediated cell adhesion FAT1 mRNA levels increase in some cell lines with increasing cell density but not in others (As proof of the utility of the lines, we studied the impact of loss of functional utations D. For exn others B, this hCASP8 and FAT1 confirmed a clonal growth advantage, consistent with the observation that CASP8 mutant cells are more aggressive in tongue xenografts CASP8 mutant cells CASP8 and FAT1 will be useful for exploring the cancer relevance of the observed antagonistic interactions between FAT1 and Caspase 8 Knockdown of each gene in cells that were wild type for In conclusion, OSCC is highly heterogeneous at both the cellular and genetic levels. The panel of cell lines that we have characterised will be useful for identifying the signalling pathways that are affected by particular combinations of mutations and for discovering how they act both at the level of the cells that bear the mutations and at the level of the surrounding stroma and immune infiltrate. In the long term, this type of approach has the potential to suggest new combination therapies to improve the outcome for patients with OSCC.


Targeting mitochondrial energy metabolism is a novel approach in cancer research and can be traced back to the description of the Warburg effect. Dichloroacetate, a controversially discussed subject of many studies in cancer research, is a pyruvate dehydrogenase kinase inhibitor. Dichloroacetate causes metabolic changes in cancerous glycolysis towards oxidative phosphorylation via indirect activation of pyruvate dehydrogenase in mitochondria. Canine mammary cancer is frequently diagnosed but after therapy prognosis still remains poor. In this study, canine mammary carcinoma, adenoma and non-neoplastic mammary gland cell lines were treated using 10 mM Dichloroacetate. The effect on cell number, lactate release and PDH expression and cell respiration was investigated. Further, the effect on apoptosis and several apoptotic proteins, proliferation, and microRNA expression was evaluated. Dichloroacetate was found to reduce cell proliferation without inducing apoptosis in all examined cell lines. Mammary tumors are the most frequent neoplasia in intact female dogs and often accompanied with high recurrence and metastasis rates . In veteThe Warburg effect was characterized in the early 1920s by Otto Warburg and describes the metabolic energy production of most cancer cells which rely on aerobic glycolysis in presence of oxygen . Hypoxiain vitro and pointed out that non-cancerous cells were not affected [Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase inhibitor  and thusaffected . Dunbar affected  but in caffected . DCA in affected  whereas affected . Higher affected  as well affected .In previous studies we investigated the effect of DCA on canine prostate adenocarcinomas and transitional cell carcinomas in vitro  and founin vitro models to gain more information of cancer independent sensitivity [For evaluation of anticancer drug efficacy in preclinical experiments cell lines represent important sitivity –26. In tThis is the first study evaluating the effect of 10 mM DCA on canine mammary carcinoma as well as canine mammary adenoma cell lines in comparison to a non-cancerous mammary gland derived cell line and a non-treated control. Therefore, the influence on cell counts, viability, apoptosis, mitochondrial respiration and proliferation was examined. Furthermore, the expression of microRNA involved in proliferation and apoptosis was determined.Four cell lines derived from different mammary tissues were used for experiments. MTH53A (non-neoplastic mammary gland), MTH52C (mammary carcinoma) and ZMTH3 (mammary adenoma) were transfected with SV-40 and routinely maintained in the Small Animal Clinic, University of Veterinary Medicine, Hannover, Germany. SV-40 transfection was performed to achieve immortalization as primary cultures derived from mammary tissue  showed limited proliferation capacity. DT14/06T (mammary carcinoma) was established by continuous cultivation in the Small Animal Clinic, University of Veterinary Medicine Hannover, Germany. The cell lines were classified after pathohistological examination of the primary tissue. All tissues used for cell line establishment were collected as part of routine mastectomy and with the owner’s permission. Therefore according to German standards, ethical approval was not required.2 culture flasks  with medium 199  containing 10% fetal calf serum , 2% penicillin-streptomycin  and incubated at 37°C and 5% CO2 in humidified atmosphere. At 85% confluency cells were trypsinized with TrypLETM Express  and were splitted 1:2.All cell lines were routinely maintained in 25 cm2 flasks . DCA was diluted in deionized water and filter-sterilized after pH modulation to 7.4 with NaOH. All cell lines were cultured without DCA and used as non-treated control respectively. The concentration of 10 mM DCA was chosen in order to allow comparability with previous human [in vitro studies. Furthermore, this dosage is applied in an explorative study setting.Cells used in experiments were exposed to 10 mM DCA over 48 hours and cultured with 10 ml medium in 75 cmus human  and canius human  in vitroTM Express  cell number was counted with an automated CellometerTM Auto T4  and compared with untreated control. Cells were washed with PBS, centrifuged at 1000 rpm for 10 minutes (Biochrom) and stored at -80°C for further examinations .After washing with PBS to remove dead cells and trypsinization with TrypLETM BCA Assay (Thermofisher Scientific) as described in manufacturer´s instructions.Lactate release in media was performed by colorimetric determination with Cobas® C311 . Therefore growth media from cell culture was centrifuged at 1000 rpm for 10 minutes to remove floating cells and debris and 1.3 ml were removed to a sodium fluoride vessel . To eliminate influences of phenol red and fetal calf serum, medium was used as negative control and deducted from measurements. Further the lactate content was normalized to intracellular protein concentration to prevent influences of cell numbers and volume. Protein concentration was assessed with Pierce2. Measurements were performed every 24 hours for four days. Therefore medium was replaced and 20 μl MTT  was added and incubated for two hours at 37°C in the dark. Absorbance was determined with a Synergy2 plate reader  and data were analyzed with Gen5TM 1.11 Software (BioTek). Absorbance was reduced by media negative control and in addition normalized to non-treated new seeded cells.Depending on each cell line 3000–6000 cells were seeded in a 96-well-plate  with 200 μl medium 199, 10% fetal calf serum, 2% penicillin-streptomycin, 10 mM DCA and incubated at 37°C and 5% humidified CO5 cells were cultured in a 6-well-plate  as described above. After 48 hours following 10 mM DCA exposure, cells were trypsinized and centrifuged together with supernatant containing non-adherent and dead cells at 1000 rpm for 6 minutes. Cell pellet was resuspended in 500 μl assay buffer and staining was performed as described in manufacturer’s protocol. 104 events were counted with BD FACScaliburTM  and CellQuestTM Pro 6.0 software (BD Biosciences). Annexin and Sytox were detected in FL-1. Data analysis was performed with FlowJo Version 10.0.8r1 . Gates were determined based on positive controls (cells permeabilized with Saponin) and negative controls of each cell line (non-treated viable cells).Apoptosis and quantity of dead cells was analyzed with Annexin FITC and Sytox labeling . Therefore 106 cells using the NucleoSpin Small RNA Kit  as described in manufacturer’s protocol. 35 ng total RNA was used for cDNA synthesis using Taqman® MicroRNA Reverse Transkription Kit  according to manufacturer´s instructions. Following cycle conditions were used: 30 minutes 16°C, 30 minutes 42°C, 5 minutes 85°C. Relative quantification of microRNA expression of treated cells in comparison to untreated control was performed with Eppendorff realplex4 Cycler  using 1.33 μl cDNA in a total volume of 20 μl containing TaqMan® Universal Master Mix NoAmpErase® UNG  and TaqMan® MicroRNA assays for Mir141 (ID 245445_mat), Mir145 (ID 002278), Mir375 (ID 000564) purchased from Thermofisher Scientific. Procedure was maintained as manufacturer´s protocol conducting following conditions: 95°C for 10 minutes subsequently 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Data were normalized to the reference gene RNU6B (ID 001093) and analysis was performed using Rest2009 . Samples with Ct-values >35 were excluded from analysis.Isolation of total RNA was performed with 10TM instrument  and processed with xPONENT 3.1 software (Luminex Corporation). Additionally, samples for PDH measurements were filtered with centrifugal ultrafree filter units with a pore size of 0.65 μm (Merck Millipore) at 7000 rpm for 4 minutes. Values with MFI < background MFI + 2 x standard deviation were excluded from analysis. Quantitation of survivin was performed with ProcartaPlex Human Survivin Simplex Kit  using cell culture supernatant as described in manufacturer´s protocol. Survivin values were normalized to protein concentration . Total PDH, PDH-P and apoptotic proteins (BAD and JNK) were analyzed with multiplex assays from Merck Millipore . For protein evaluation a total protein amount of 10 ng (PDH-P) or rather 25 ng (apoptotic proteins JNK and BAD) was used, which was determined with Pierce BCA Assay according to manufacturer’s instruction (Thermofisher Scientific).Samples for protein expression analysis were prepared as specified in manufacturer´s protocol and measurements were performed with xMAP® Luminex Bead Technology using a Luminex 200Mitochondrial ROS production as indirect marker for mitochondrial activity was determined by staining of mitochondrial derived ROS using 4 μM MitoSox  for 15 minutes. Cells were grown on 8-well μ-dishes  treated with 10 mM DCA for 48 hours. After fixation with 4% paraformaldehyde, μ-slides were washed with Hank´s Balanced Salt Solution (HBSS) containing calcium and magnesium. After staining cell nuclei were labeled with DAPI  for 5 minutes. Fluorescence imaging was performed with an inverted confocal laser scanning microscope  using a 60x water immersion objective (Nikon). Images were taken with EZ-C1 1.80 software (Nikon). The excitation occurred with a diode laser at 408 nm (DAPI) and with a helium/neon laser at 543 nm (MitoSox). Total cell fluorescence of MitoSox deducting background was analyzed with ImageJ and normalized to cell counts.2. After 48 hours incubation 100 μl ATP-solution  was added. Following 10 minutes incubation at room temperature, luminescence measurement was performed with Synergy2 Plate Reader (BioTek). In order to normalize the data with the respective cell numbers, cells were counted in a simultaneously performed experiment by flow cytometry .Mitochondrial respiration was directly shown by measuring cellular ATP synthesis. The cell lines were seeded (5000 cells/well) as triplicate in a 96-opaque-well plate (Greiner Bio-One) with 100 μl medium 199, 10% fetal calf serum, 2% streptomycin-penicillin, 10 mM DCA and incubated at 37°C humidified COCultivation, fixation and washing were performed as described above. After permeabilization using 0.2% Triton X-100 for 20 minutes, cells were exposed overnight to a canine specific rabbit-polyclonal Ki67 antibody . For labeling, a monoclonal anti-rabbit Alexa Fluor® 555 antibody  was incubated for 1 hour (1:250) and cells were counterstained with DAPI (1:1000) for 5 minutes. Fluorescence imaging protocol was the same as described above. Total cell fluorescence was established as described above. For TUNEL staining Apoptag Fluorescein Direct Kit (Merck Millipore) was performed as manufacturer´s instructions. The excitation occurred with an argonlaser at 488 nm and imaging was performed as described above. The amount of TUNEL positive cells was evaluated.Protein expression analysis was performed with Immunoblotting (Western Blot) as described in detail by Wagner et al. . The proStatistical analysis of data was performed with SAS software 7.1 . For comparison of two means, two-tailed t-test was used. The confidence value was set to 5% (p<0.05) and was considered statistically significant.Following 48 hours DCA treatment (10 mM) the cell numbers of both mammary carcinoma cell lines MTH52C (p = 0.0171) and DT14/06T (p<0.0001) decreased significantly in comparison to non-treated control. A comparable significant effect was observed in the mammary adenoma cell line ZMTH3 (p = 0.0131) and could also be constituted in the non-cancerous mammary gland derived cell line MTH53A (p = 0.0039). Although MTH53A showed decreasing cell numbers, a statistically significant difference between the mammary carcinoma cell line DT14/06T (p = 0.0140) and the non-cancerous cell line MTH53A was present. No difference was observed between MTH53A and the adenoma derived cell line ZMTH3 or the mammary carcinoma derived cell line MTH52C .The proliferation, assessed by detection of the proliferation marker Ki67, decreased significantly in all mammary cell lines with exception of the non-neoplastic mammary gland derived cell line MTH53A in comparison to non-treated control and further a significant decrease in proliferation can be observed within comparison of MTH53A and the other cell lines. No difference was assessed between neoplastic cell lines .In comparison to negative control DCA reduced the lactate release into the media in all examined cell lines except in the benign mammary adenoma cell line ZMTH3 (p = 0.1218). No statistical difference was observed within non-cancerous and neoplastic cell lines .In order to assess the effect of DCA on PDK expression, protein analysis of PDK-1 and PDK-3 was performed. As shown in 232 decreased significantly in all cell lines whereas PDH-P at Ser293 was reduced only in the mammary carcinoma DT14/06T (p = 0.0445) (293 in comparison to untreated control. With consideration of PDH-P at Ser300 a significant decrease is apparent between mammary carcinoma derived cell lines MTH52C (p = 0.0263), DT14/06T (p = 0.0282) and non-treated control. ZMTH3 showed visible tendency of PDH-P reduction but did not reach significance (p = 0.0852). No reduction was apparent in all PDH-P residues between MTH53A and neoplastic cell lines.Phosphorylated PDH (PDH-P) and thus inactive PDH was measured with Luminex Magnetic Bead technology to evaluate whether DCA has an effect on pyruvate oxidation. After normalization to total PDH expression phosphorylated PDH at Ser 0.0445) . The mamMitochondrial respiration was evaluated via indirect measurement of mitochondrial derived ROS. As shown in After 48 hours DCA treatment, viability analysis with flow cytometry revealed no changes in apoptosis and amount of dead cells in MTH53A, ZMTH3 and DT14/06T . In MTH5To determine if trypsinization caused negative effects, apoptosis was evaluated with TUNEL method via fluorescence microscopy. No difference in number of TUNEL positive cells was evaluated in any of these cell lines .Further, two proteins  involved in apoptosis were evaluated. The protein expression was performed with Luminex Magnetic Bead technology. After 48 hours of DCA treatment, the phosphorylated and thus activated c-jun N-terminated kinases (JNK) decreased significantly in MTH53A (p = 0.0046), ZMTH3 (p = 0.0228) and MTH52C (p = 0.0131). The inverse case was observed in mammary carcinoma cell line DT14/06T (p = 0.0121) presenting significantly increased active JNK expression .The phosphorylated and thus inactive form of Bcl-2-Antagonist-of-cell-death (BAD) showed significantly increased values in all neoplastic cell lines. In MTH53A (p = 0.2340) no difference was detectable .Decreased survivin expression is apparent in all cell lines but significance is present in cell line DT14/06T (p = 0.0125) in comparison to untreated control as well as in comparison to non-neoplastic mammary gland derived cell line MTH53A (p = 0.0200) .The metabolic activity as reference for proliferation and viability was assessed during 96 hours ongoing DCA treatment . After 9Three microRNAs involved in apoptosis and proliferation were analyzed with quantitative RT-PCR. miR-145 was detectable only in the mammary carcinoma cell line DT14/06T  and showThe initial aim of this study was to determine which metabolic processes occur in canine mammary gland derived cell lines during DCA exposure and if DCA might be beneficial for cancer treatment in dogs bearing mammary gland neoplasia. The results presented illustrate that DCA has anti-proliferative effects on cell growth which is shown in decreased cell number and reduction of the proliferation marker Ki67 expression. The growth inhibiting effect was likewise observed in several human studies –32 incluDue to increased cellular respiration and thus mitochondrial ROS production, it is hypothesized that DCA induces mitochondrial dependent apoptosis by release of cytochrome c and activation of several apoptosis proteins . HoweverApart from this, survivin expression, an apoptosis inhibitor and proliferation promotor , 43, sho232 and Ser300 appear to be the most important metabolic modulation in canine PDH regulation. This supports the assumption of Rardin et al., that PDH phosphorylation at Ser232 is not limited to heart tissue [293 phosphorylation was not significantly affected by DCA [293 is reported to be phosphorylated rapidly by PDK, especially isoform 2 [DCA, a known PDK inhibitor, shifted the energy production from glycolysis towards normal cellular respiration with glucose oxidation . This ist tissue . In contsoform 2 , indicatIn order to evaluate the effect of DCA on cell division and apoptosis, miR-141, miR-145 and miR-375, often deregulated in cancer , were anIn comparison to human mammary cancer cell lines following DCA treatment, canine mammary cell lines are almost consistent , 41. DCAin vivo without severe side effects. But these data indicate that DCA might be supportive in sensitization of cancer cells to other chemotherapeutics [Further the dose of 10 mM DCA is clinically not achievable apeutics . Furtherapeutics .S1 FigIn comparison to non-treated control, no changes in apoptosis were observed. Data are shown as mean ± SD; n = 3 and are presented as percentage of TUNEL positive cells in comparison to negative control. Statistical analysis was performed with two-tailed t-test; *p>0.05.(TIFF)Click here for additional data file.S2 FigWestern Blot analysis of PDK-3 expression in MTH53A, MTH52C, ZMTH3 and DT14/06T. PDK-3 expression was not detectable in all cell lines. GAPDH and α-tubulin were used as loading control.(TIFF)Click here for additional data file.