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

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

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

<i>Bi-L-Dek_K5-tTA</i> mice express luciferase and overexpress Dek in stratified squamous epithelium.

<p>(<b>A</b>) <i>In vivo</i> imaging system (IVIS) analysis depicts a single (<i>Bi-L-Dek</i>) and a bi-transgenic (<i>Bi-L-Dek_K5-tTA)</i> mouse after intraperitoneal injection of luciferin for luciferase detection in the skin of <i>Bi-L-Dek_K5-tTA</i> mice. (<b>B</b>) <i>Ex vivo</i> IVIS analysis of single transgenic (<i>K5-tTA</i>) versus bi-transgenic <i>(Bi-L-Dek_K5-tTA)</i> flank skin, ear, and esophagus following injection of luciferin, sacrifice, and dissection. (<b>C</b>) RT- qPCR of Dek mRNA levels in skin epithelium obtained from the flank of mice show a 3 fold induction of Dek transcript levels that is repressed to endogenous levels after seven days on dox chow. Primers detect endogenous and exogenous <i>Dek</i>. Error bars represent three mice for each genotype excluding the <i>Dek-/-</i> negative control which represents one mouse repeated in triplicate. (<b>D</b>) Representative western blot analysis for the detection of Dek protein levels in flank skin epithelium demonstrates increased levels of Dek protein in <i>Bi-L-Dek_K5-tTA</i> mice over those on dox and single transgenic controls. (<b>E</b>) Immunohistochemistry (IHC) with DEK antibodies (BD Biosciences, San Jose, CA, USA) in tongue epithelium confirms Dek protein overexpression in <i>Bi-L-Dek_K5-tTA</i> mice that is repressed within seven days of dox chow. (<b>F</b>) Immunofluorescence (IF) of cultured skin keratinocytes isolated from newborn <i>Bi-L-Dek_K5-tTA</i> pups with or without dox and their single transgenic littermates. Dox treated keratinocytes were cultured with 1ug/ml of dox for 48 hours before fixation. IF images of keratinocytes were taken at the same magnification and exposure after being probed for Dek, keratin 5 (K5), and stained with DAPI. (<b>G</b>) The mean fluorescent intensity of Dek staining in <b>2F</b> was quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007227#pgen.1007227.ref089" target="_blank">89</a>].</p

Dek overexpression increases the incidence of gross esophageal tumors.

<p>(<b>A</b>) Details on mice and pathologies including esophageal tumor volumes in the 4NQO-treated mice. (<b>B</b>) Representative IHC images for Dek protein overexpression in the esophagus of <i>Bi-L-Dek_K5-tTA</i> mice treated with 4NQO compared to mice on dox (Dek antibody: Cusabio, Balitmore, MD, USA; magnification: 40x). (<b>C</b>) Percent incidence of gross, microscopic, invasive, and multifocal tumors within the two groups of mice. Statistics is indicated when significantly different between the no dox/dox treated groups as determined by a Fisher Exact test. (<b>D</b>) Gross tumor volumes within the two groups. Each dot represents total gross tumor volume per mouse (no statistics due to an n = 1 for the no dox group). (<b>E</b>) Survival of the <i>Bi-L-Dek_K5-tTA</i> Dek overexpressing mice +/- dox treatment. Tissue from a seventh <i>Bi-L-Dek_K5-tTA</i> mouse that died at 27 weeks could not be evaluated for tumors at necropsy (not included in Fig 5A). (<b>F-G</b>) Images of esophagi at the time of dissection (top), and the corresponding H&E stained histologic sections of esophagus (middle), and Dek staining by IHC in the corresponding tumor (bottom) from <i>Bi-L-Dek_K5-tTA</i> mice in the absence (<b>F</b>) or presence (<b>G</b>) of dox (H&E magnification: 2x; Dek IHC magnification: 120x; Dek antibody: Cusabio, Baltimore, MD, USA). (<b>H-I</b>) Images of H&E stained esophageal sections illustrate morphological features of tumors in (<b>H</b>) Dek overexpressing <i>Bi-L-Dek_K5-tTA</i> mice and (<b>I</b>) normal esophagus and tumors in dox treated <i>Bi-L-Dek_K5-tTA</i> mice. Extensive necrosis in a poorly differentiated invasive squamous cell carcinoma (H, left panel arrows), and dyskeratotic cells (H, middle panel arrows) along with cellular dysplasia and intercellular bridges (H, middle panel inset), and extensive stromal invasion (H, right panel arrows) with focal squamous differentiation (H, right panel arrowhead) in papillary squamous cell carcinoma in Dek overexpressing mice are shown. Esophageal images from dox treated <i>Bi-L-Dek_K5-tTA</i> mice illustrate the normal esophagus from mouse lacking tumors (I, left panel), a microscopic papillary squamous cell carcinoma with minimal superficial stroma invasion (I, middle panel, arrows), and the single grossly apparent tumor characterized as a well differentiated invasive squamous cell carcinoma with abundant keratin production (I, right panel, arrows and inset). (Original magnifications: 40x, inserts 100x).</p

<i>Bi-L-Dek</i> transgene expression is detected in the context of Dek knockout mice.

<p>(<b>A</b>) <i>Bi-L-Dek_K5-tTA</i> mice were bred to Dek knockout (<i>Dek-/-)</i> mice to quantify Dek expression in the absence of endogenous Dek protein. (<b>B</b>) IVIS image of <i>Dek-/-</i> _<i>Bi-L-Dek_K5-tTA</i> mice with luciferase expression compared to <i>Dek-/-</i> and single transgenic <i>K5-tTA</i> mice after luciferin injection. (<b>C</b>) Western blot analysis detects Dek protein expression in murine flank skin from <i>Dek-/-</i> _<i>Bi-L-Dek_K5-tTA</i> mice.</p