DEK over-expression promotes mitotic defects and micronucleus formation

<p>The <i>DEK</i> gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation</p

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth

DEK overexpression in HNSCC cells increases glycolytic end products and TCA cycle intermediates.

<p>(A-B) PCA scores plot of C-SCC1 generated from normalized bucket intensities showing separation by metabolite presence between C-SCC1 R780 and R-DEK cells (A) and media (B). (C-D) Fold change in bucket intensities for each significantly changed metabolite between R780 and R-DEK cells (C) and media (D) arranged by magnitude of change. Error bars represent the SEM in fold change of the R780-DEK samples relative to the mean of the R780 controls. (E-F) The bucket intensity of metabolites in the unconditioned media (dashed red line) were compared to R780 (grey) and R-DEK (black) conditioned media samples to identify metabolites decreased (E) and those increased compared to control unconditioned media (F). (G) Metabolic pathway analysis highlighting metabolites identified by NMR that are differently regulated upon DEK overexpression. The metabolites identified are involved in various metabolic pathways including choline metabolism, protein and nucleotide synthesis, cellular redox state, aerobic glycolysis, and the TCA cycle. Abbreviations: p = phospho, Asn = asparagine, Phe = phenylalanine, GPC = glycerophosphocholine, NAD⁺ = nicotinamide adenine dinucleotide, Myo-ino = myo-inositol, Glu = glutamate, Gln = glutamine, α-keto = α-ketoglutarate, 2-OIC = 2-oxoisocaproate, Poly-Glu = polyglutamate, DMA = dimethylamine.</p

DEK overexpression increases glycolysis and the maximum rate of oxidative phosphorylation in NIKS and C-SCC1 cells.

<p>Seahorse XF24 Extracellular Flux Analyzer experiments using the mitochondrial stress test. (A-D) Quantification of oxygen consumption rate (OCR) measurements from 4 replicates of NIKS (A) and C-SCC1 (C) R780 and R-DEK samples taken three times at baseline and after treatment with the following pharmacological inhibitors of metabolism: oligomycin (ATP synthetase inhibitor), FCCP (and uncoupling agent), and rotenone and antimycin A (electron transport chain inhibitors). (B and D) Calculations from the mitochondrial stress test were as follows: non-mitochondrial respiration = oxygen consumed after treatment with electron transport chain inhibitors (rotenone and antimycin A). Basal OCR = baseline OCR minus non-mitochondrial respiration. ATP production = baseline OCR minus OCR after ATP synthetase inhibitor (oligomycin). Spare capacity = max OCR minus baseline OCR. Proton leak = OCR after oligomycin treatment minus OCR with electron transport chain inhibitors (rotenone and antimycin A). (E-H) The extracellular acidification rate (ECAR) was quantified for NIKS (E-F) and C-SCC1 (G-H) transduced with R780 or R-DEK. Quantification of glycolysis was calculated for baseline, maximum potential, and reserve potential in NIKS (F) and C-SCC1 (H). Reserve ECAR was calculated by subtracting baseline ECAR from maximum ECAR (oligomycin treated). Error bars represent the SEM of the 4 replicates. Statistical significance was determined using a t-test. Where indicated *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, and ***<i>P</i> ≤ 0.001.</p

DEK overexpression drives glycolytic and glutathione pathways in NIKS and C-SCC1 cells, but uniquely stimulations TCA cycle intermediate accumulation in C-SCC1 cells.

<p>(A) Fold change in R-DEK compared to R780 bucket intensities for metabolites identified in NIKS (grey bars) and C-SCC1 cells (black bars). Metabolite names labelled in blue are decreased and those in red are increased in both cell lines. Metabolites labelled in green are either jointly but differentially regulated or only regulated in one cell line; in either case, the metabolite is higher in the C-SCC1 cells and/or lower in the NIKS with DEK overexpression. (B-E) Metabolites regulated in one or both normal/cancer cells are indicated within known associated metabolic pathways. (B) Metabolite products of aerobic glycolysis are increased in both cell lines (red) with an increase in glucose uptake in the R-DEK NIKS. (C) Glutathione and aspartate are decreased in both cell lines while metabolites surrounding this pathway are decreased in NIKS (green) and increased in C-SCC1 cells. (D) Choline and p-choline are decreased in both cell lines while GPC was differentially regulated. (E) Metabolites in and surrounding the TCA cycle are increased in the C-SCC1 cells (green) and either unchanged or decreased in the NIKS. Abbreviations: p = phospho, GPC = glycerophosphocholine, NAD+ = nicotinamide adenine dinucleotide, α-keto = α-ketoglutarate, OAA = oxaloacetate, ROS = reactive oxygen species.</p

DEK overexpression increases the metabolic end products of glycolysis and the utilization of amino acids in keratinocytes.

<p>(A-B) Principal components analysis (PCA) scores plots of NIKS generated from normalized bucket intensities for 8 replicates showing separation based on metabolite presence between NIKS R780 (grey) and R-DEK (black) cells (A) and in their respective conditioned media (B). (C-D) Fold change in bucket intensities for each metabolite that was significantly different between R780 and R-DEK cells (C) and in their respective conditioned media (D) is arranged by magnitude of change. Metabolites in red are increased by DEK overexpression and metabolites in blue are decreased by DEK overexpression. Error bars represent the SEM in fold change of the 8 R780-DEK samples relative to the mean of the R780 controls. (E-F) The bucket intensity of metabolites averaged from triplicate samples of unconditioned media (dashed red line) were compared to R780 (grey) and R-DEK (black) conditioned media samples to identify metabolites that are decreased (E) and increased (F) compared to unconditioned control media. (G) Metabolic pathway schematic highlighting metabolites identified by NMR which were differentially regulated by DEK overexpression. The pathway analysis reveals many of metabolites increased upon DEK expression are products of aerobic glycolysis. Abbreviations: 1-MNA = 1 methylnicotinamide, p = phospho, Asn = asparagine, Phe = phenylalanine, GPC = glycerophosphocholine, NAD⁺ = nicotinamide adenine dinucleotide, Myo-ino = myo-inositol, Glu = glutamate, Gln = glutamine, α-keto = α-ketoglutarate.</p

DEK overexpression increases cellular metabolic activity in NIKS and C-SCC1 cells in the absence of proliferative gains.

<p>(A-B) Western blot analysis validates DEK overexpression in NIKS (A) and C-SCC1 (B) cells with accompanying phase/light microscopy images taken at 10x magnification with a 20x inset of cells transduced with retroviral vector R780 control or R780-DEK (R-DEK). Images were taken on day 2 post plating. (C-D) Equal numbers of NIKS (C) and C-SCC1 (D) R780 or R-DEK cells were plated and counted over 4 days in 4 independent experiments. (E-F) Cell cycle profiles quantified by flow cytometry in NIKS (E) and C-SCC1 (F) R780 and R-DEK cells pulsed with EdU for 2 hours and stained with propidium iodide. The percentage of cells in each phase of the cell cycle was quantified from triplicate wells in three independent experiments. (G-H) Flow cytometry analysis of cleaved caspase 3 for the detection of apoptosis in NIKS (G) and C-SCC1 (H) R780 versus R-DEK cells. (I-J) Fold change in absorbance at 490 nm for MTS assay from 10,000 NIKS (I) or C-SCC1 (J) cells plated in a 96-well plate and measured 24 hours post plating at ~80% confluency. All error bars represent the standard error of the mean (SEM).</p

Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence

<div><p>Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression <i>in vivo</i>, we generated and validated a tetracycline responsive <i>Dek</i> transgenic mouse model referred to as <i>Bi-L-Dek</i>. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing <i>Bi-L-Dek</i> mice to keratin 5 tetracycline transactivator (<i>K5-tTA</i>) mice. Conditional transgene expression was validated in the resulting <i>Bi-L-Dek_K5-tTA</i> mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these data demonstrate that Dek overexpression in the cell of origin for SCC is sufficient to promote esophageal SCC development <i>in vivo</i>.</p></div

Generation of a tetracycline off <i>Dek</i> transgenic mouse model.

<p>(<b>A</b>) <i>Bi-L-Dek</i> transgenic mice were engineered by micronuclear injection of linearized <i>Bi-L-Dek</i> DNA into the pronucleus of FVB/N fertilized eggs. <i>Bi-L-Dek</i> mice harbor a tetracycline response element (TRE) that controls two mini cytomegalovirus (CMV) promoters driving bi-directional transcription of <i>Dek</i> and <i>luciferase</i>. (<b>B</b>) Copy number analysis of the <i>Bi-L-Dek</i> transgene in founder #317 identified 2–4 insertions in the F2-F4 generation. Error bars represent differences between 2–3 mice for each generation excluding F0 for which only one mouse exists. F3 and subsequent generations from this founder line were used for the experiments. <b>(C)</b> <i>Bi-L-Dek</i> mice were bred to keratin 5 promoter driven tetracycline transactivator (<i>K5-tTA)</i> mice. (<b>D</b>) <i>Bi-L-Dek</i> and <i>K5-tTA</i> transgene presence in offspring was confirmed by genotyping along with identification of single transgenic and non-transgenic (Non Tg) littermates. FVB/N (WT) mice were negative controls (-) and the F2 parent carrying the transgene was the positive control (+). (<b>E</b>) Schematic of <i>Bi-L-Dek_K5-tTA</i> mice designed to express luciferase and to overexpress Dek in the K5-positive basal layer of stratified squamous epithelium (highlighted in blue). Transgene repression by dox in this tet-off system is indicated.</p