FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation

The E3 ubiquitin ligase component FBXW7 modulates homeostasis and inhibits tumorigenesis in the murine intestine

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