Scientific Papers

Aspirin reprogrammes colorectal cancer cell metabolism and sensitises to glutaminase inhibition | Cancer & Metabolism


Cell lines and culture

The human colorectal carcinoma-derived cell lines; SW620 and LS174T were obtained from the American Type Culture Collection (ATCC, Maryland, USA), and HCA7 was a kind gift from Dr. Susan Kirkland, Imperial College, London. All cell lines were routinely tested for mycoplasma contamination using MycoAlert PLUS mycoplasma detection kit (Lonza, MD, USA) and molecularly characterised using an “in house” panel of cellular and molecular markers to check that cell lines have not been cross contaminated (every 3–6 months). Stocks were securely catalogued and stored; passage numbers strictly adhered to prevent phenotypic drift. All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, Merck, KGAa) with added 10% foetal bovine serum (FBS) (Sigma-Aldrich, Merck, KGaA), 2 mM glutamine (Gibco, ThermoFisher Scientific Inc.), 100 units/ml penicillin and 100 units/ml streptomycin (Gibco, ThermoFisher Scientific Inc). For stock purposes, cells were maintained in 25cm2 tissue culture (T25) flasks (Corning, NY, USA) and incubated at 37℃ in dry incubators maintained at 5% CO2. Cell media were changed every 3–4 days. Experiments were performed in triplicate independently with distinct passages of cells, unless otherwise stated.

Treatments

Long-term aspirin

For the long-term aspirin-treated cells, a 20-mM stock solution of aspirin (Sigma, Merck KGaA, Darmstadt, Germany) was created by adding 3.6 mg/ml aspirin to 10% DMEM, and fresh aspirin was made up immediately prior to use. Aspirin concentration in the growth media was maintained continuously for ~ 52 weeks. Passage frequency and ratio were adjusted to maintain confluency in aspirin-treated cells.

CB-839, inhibitor 968 and UK-5099

Stock of CB-839 (Sigma-Aldrich, Merck, KGaA), inhibitor 968 (Sigma-Aldrich, Merck, KGaA) and UK-5099 (Sigma Aldrich, Merck, KGaA) were made in dimethyl sulfoxide (DMSO). DMSO concentration was maintained consistently in all treatment concentrations and vehicle control.

Proliferation assays

Crystal violet staining

To measure the proliferation of cells treated with aspirin in combination with either CB-839 or UK-5099, cells were seeded into 96 well plates (Corning, NY, USA) (20,000 cells per well in all conditions except HCA7 cells treated with 4 mM aspirin, where 40,000 cells per well were seeded) in normal growth medium and incubated for 24 h, with 3–4 technical replicate wells per treatment condition. Cells were then treated with media containing drug treatments (or vehicle control) and incubated for a further 72 h. Plates were then fixed with 4% PFA for 15 min, then stained with 0.5% crystal violet solution (Sigma-Aldrich, Merck KGaA), before solubilisation in 2% SDS, and subsequent OD595 measurements were obtained using an iMark microplate reader (Bio-Rad, Laboratories, Inc.). The number of adherent cells was claculated by the confluence in each concentration of CB-839/UK-5099 at 72 h, relative to the same concentration of aspirin in control conditions, in order to compare the effect of the drugs between different aspirin treatments.

For experiments using Human Plasma-Like Medium (HPLM – Gibco, ThermoFisher Scientific, Inc.), HPLM was supplemented with 10% dialysed FBS (dFBS) and experiments were performed as above. Cells were incubated in 10% dFBS HPLM for at least 48 h prior to the start of the treatment, to allow for metabolic adaptation. During the experiment, media was changed every 24 h (unlike experiments in DMEM where the same media was left for the full 72 h of treatment), in order to avoid depletion of the low levels of nutrients.

IncuCyte

To simultaneously measure cell proliferation and apoptosis upon treatment with aspirin in combination with CB-839, a IncuCyte ZOOM live cell imaging system was used. Cells were seeded in 96 well plates (20,000 cells per well) and incubated for 24 h, with 3–4 wells per treatment condition (technical replicates). Cells were then treated with treatment-containing media (or vehicle control) and placed in the IncuCyte system. The percentage of confluence was measured every 4 h for the total time indicated on the graphs. The IncuCyte system took four different image fields per well. At the time of treatment, the cells were also treated with 2-µM CellEvent caspase-3/7 green detection reagent (C10423; Invitrogen, ThermoFisher Scientific, Inc), which was used to measure apoptosis. Green fluorescent cells, indicating active caspase-3/7 and apoptosis, were measured by the IncuCyte system as green object count (1/mm2). For each individual well, the green object count was normalised to the confluence at each timepoint, and results were expressed as relative apoptosis. The same method was performed using SW620 cells treated with 2 µM ABT-737 compared to a vehicle control prior to the start of the assay, as a positive control for apoptosis in order to validate this assay and confirm the detection of apoptotic cells.

Proteomic analysis

TMT labelling and high pH reversed-phase chromatography

Following 52 weeks of aspirin treatment to develop long-term treated cell lines, cells were seeded in T25 flasks, and following maintenance in aspirin for a further 72 h whole-cell protein lysates were collected. Lysates were collected as described previously [19]. Protein concentrations were ascertained, and samples were adjusted to 2 mg/mL. One hundred micrograms of each sample was digested with trypsin overnight at 37℃, labelled with tandem mass tag (TMT) ten plex reagents according to the manufacturer’s protocol (ThermoFisher Scientific, Inc.) and the labelled samples pooled.

An aliquot of the pooled sample was evaporated to dryness, resuspended in 5% formic acid and then desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was again evaporated to dryness and resuspended in buffer A (20-mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an ultimate 3000 liquid chromatography system (ThermoFisher Scientific, Inc.). In brief, the sample was loaded onto a XBridge BEH C18 column (130 Å, 3.5 µm, 2.1 mm X 150 mm, Waters, Milford, Massachusetts, USA) in buffer A and peptides eluted with an increasing gradient of buffer B (20-mM ammonium hydroxide in acetonitrile, pH 10) from 0 to 95% over 60 min. The resulting fractions (15 in total) were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher Scientific, Inc.).

Nano-LC mass spectrometry

High pH RP fractions were further fractionated using an ultimate 3000 nano-LC system in line with an Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher Scientific, Inc.). In brief, peptides in 1% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (ThermoFisher Scientific, Inc.). After washing with 0.5% (v/v), acetonitrile 0.1% (v/v) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse-phase analytical column (ThermoFisher Scientific, Inc.) over a 150-min organic gradient, using 7 gradient segments (1–6% solvent B over 1 min, 6–15% B over 58 min, 15–32% B over 58 min, 32–40%B over 5 min, 40–90%B over 1 min, held at 90%B for 6 min and then reduced to 1%B over 1 min) with a flow rate of 300 nl min−1. Solvent A was 0.1% formic acid, and solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionised by nano-electrospray ionisation at 2.0 kV using a stainless-steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary temperature of 275℃.

All spectra were acquired using an Orbitrap Fusion Tribrid mass spectrometer controlled by Xcalibur 2.1 software (Thermo Scientific) and operated in data-dependent acquisition mode using an SPS-MS3 workflow. FTMS1 spectra were collected at a resolution of 120,000, with an automatic gain control (AGC) target of 200,000 and a max injection time of 50 ms. Precursors were filtered with an intensity threshold of 5000, according to charge state (to include charge states 2–7) and with monoisotopic peak determination set to peptide. Previously interrogated precursors were excluded using a dynamic window (60 s ± 10 ppm). The MS2 precursors were isolated with a quadrupole isolation window of 1.2 m/z. ITMS2 spectra were collected with an AGC target of 10,000, max injection time of 70 ms and CID collision energy of 35%.

For FTMS3 analysis, the Orbitrap was operated at 50,000 resolution with an AGC target of 50,000 and a max injection time of 105 ms. Precursors were fragmented by high energy collision dissociation (HCD) at a normalised collision energy of 60% to ensure maximal TMT reporter ion yield. Synchronous precursor selection (SPS) was enabled to include up to 5 MS2 fragment ions in the FTMS3 scan.

Data analysis

The raw data files (supplied in Supplementary Data File S2) were processed and quantified using Proteome Discoverer software v2.1 (ThermoFisher Scientific, Inc.) and searched against the UniProt Human database (downloaded September 2017; 140,000 sequences) using the SEQUEST HT algorithm. Peptide precursor mass tolerance was set at 10 ppm, and MS/MS tolerance was set at 0.6 Da. Search criteria included oxidation of methionine (+ 15.995 Da), acetylation of the protein N terminus (+ 42.011 Da) and methionine loss plus acetylation of the protein N terminus (− 89.03 Da) as variable modifications and carbamidomethylation of cysteine (+ 57.021 Da) and the addition of the TMT mass tag (+ 229.163 Da) to peptide N termini and lysine as fixed modifications. Searches were performed with full tryptic digestion, and a maximum of two missed cleavages were allowed. The reverse database search option was enabled, and all data was filtered to satisfy a false discovery rate (FDR) of 5%.

Protein abundance processing

Protein groupings were determined by PD2.1; however, the master protein selection was improved with an in-house script. This enables us to infer biological trends more effectively in the dataset without any loss in the quality of identification or quantification. The MS data were searched against the human Uniprot database retrieved on October 2, 2019, and updated with additional annotation information on April 21, 2020.

The protein abundances were normalised within each sample to the total peptide amount, then Log2 transformed to bring them closer to a normal distribution.

Statistics

Statistical significance was then determined using Welch’s T tests between the conditions of interest. The p values were FDR-corrected using the Benjamini–Hochberg method.

QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA)

Data were analysed using ingenuity pathway analysis. Proteins from the dataset that met the cutoff of p < 0.05 were considered for the analysis and compared to a reference set consisting of the full list of proteins identified in the experiment. A right-tailed Fisher’s exact test was used to calculate a p value determining the probability that the association between the genes in the dataset and the pathways/upstream regulators/functions was by chance alone, and the predicted and observed regulation patterns of the proteins were used to predict an activation z score.

Overrepresentation analysis

Overrepresentation analysis was performed using Webgestalt (www.webgestalt.org), using the functional databases Geneontology, and pathways (KEGG—Kyoto Encyclopedia of Genes and Genomes). Gene symbols were entered for proteins that showed signification regulation (p < 0.05, fold change > 1.4 or < 0.71), in both 2-mM and 4-mM long-term aspirin compared to control.

SDS-PAGE and immunoblotting

Cell lysates were prepared and subjected to western analysis as described previously [19] using antibodies to the following: α-tubulin (T9026, Sigma-Aldrich, Merck, Inc.), ATF4 (11,815, Cell Signaling Technology Inc. (CST)), ASCT2 (5345, CST), GLS1 (88,964, CST), GPT2 (16,757–1-AP, ProteinTech, Group, Inc.), LAT1 (5347, CST), PC (ab126707, Abcam, Cambridge, UK), PDK1 (3820, CST), HK1 (2024, CST), and GLUT1 (21,829–1-AP20, Proteintech, Group, Inc.). All were used at 1:1000 dilution, except for α-tubulin which was used at 1:10,000 dilution. The density of bands detected by immunoblotting was measured using ImageJ. In order to compare data from independent experiments, protein expression changes were calculated as relative changes from the control conditions in each replicate. Results from at least three independent experiments were analysed with this method.

Quantitative reverse transcriptase-PCR (qPCR)

Total RNA was extracted using Tri-Reagent (Sigma-Aldrich) as per manufacturer’s instruction an RNAeasy mini kit with an ON-column DNA digest step (Qiagen, Limberg, Netherlands) was used according to the manufacturer’s instructions to clean up the RNA. RNA concentration and purity were measured using a NanoDrop™ spectrophotometer (ThermoFisher Scientific Inc.). Synthesis of cDNA and qRT-PCR were performed as previously described [20] using the following primers (all from Qiagen, Limberg, Netherlands): ATF4 (QT00074466), GPT2 (QT00066381), GLS1 (QT00019397), PC (QT01005592), PDK1 (QT00069636), SLC7A11 (QT00002674) and SLC7A5 (QT00089145). Gene expression was normalised to the housekeeping genes TBP (cat. no. QT00000721) or HPRT (QT00059066), both from Qiagen.

Extracellular flux analysis

Extracellular flux analysis was carried out using the XFp Seahorse Extracellular Flux Analyzer (Agilent), according to the manufacturer’s protocol. Long-term aspirin-treated SW620 cells (60,000 cells) were seeded onto a Cell-Tak (354,240, Corning, NY, USA)-coated microplate (2–3 technical replicate wells per condition) and centrifuged at 200 g for 1 min (no brake), allowing for immediate adhesion. Cells were seeded in Seahorse XF assay media (Agilent, CA, USA) supplemented with 10 mM glucose, 2 mM glutamine and 1 mM pyruvate (Agilent, CA, USA). Corresponding OCR/ECAR (oxygen consumption rate/extracellular acidification rate) changes were monitored for the duration of the experiment. Wells had subsequent injections of oligomycin (2 µM), FCCP (2 µM), antimycin A (1 µM) and rotenone (1 µM) and monensin (20 µM) (in order to determine the maximal glycolytic rate, as shown by Mookerjee et al. [21, 22]) all from Sigma-Aldrich. Data were acquired using the Seahorse Wave software v2.6 (Agilent, CA, USA). The experiment was performed independently in triplicate.

Stable isotope tracer analysis

For stable isotope labelling (SIL) experiments, cells were cultured with U-[13C]-Glc or U-[13C]-Q (Cambridge Isotopes Laboratories, Inc.) for the indicated time points. 13C-labelled nutrients were added to glucose, glutamine-free DMEM, supplemented with 10% dFBS, 100 units/ml penicillin and 100 units/ml streptomycin, 10 mM glucose and 2 mM glutamine (13C-labelled or unlabelled as appropriate). Cellular metabolites were extracted and analysed by gas chromatography-mass spectrometry (GC–MS) using protocols described previously [23,24,25]. Metabolite extracts were derived using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) as described previously [26]. D-myristic acid (750 ng/sample) was added as an internal standard to metabolite extracts, and metabolite abundance was expressed relative to the internal standard and normalised to cell number. Mass isotopomer distribution was determined using a custom algorithm developed at McGill University [25]. The experiment was performed with three different flasks of cells from the same passage number per condition. Raw data are supplied in Supplementary Data File S1.

In vivo experiments

All in vivo experiments were carried out in accordance with the UK Home Office regulations (under project licences: 70/8646 and PP3908577) and by adhering to the ARRIVE guidelines with approval from the Animal Welfare and Ethical Review Board of the University of Glasgow. Mice were housed under a 12-h light–dark cycle, at constant temperature (19–23℃) and humidity (55 ± 10%). Standard diet and water were available ad libitum. The majority of the work was performed in the C57BL/6J background. The following alleles were used in this study: VillinCreER [27], Apcfl [28]. Full intestinal recombination was obtained by two intraperitoneal injections of 2mg tamoxifen, and tissues were harvested 4 days post induction. For drug studies in vivo, Villin-CreERT2Apcfl/fl mice were treated with CB-839 (200mg/kg in 25% (w/v) hydroxypropyl-β-cyclodextrin in 10mm citrate at pH 2.0) or vehicle from day 1 post i.p. tamoxifen administration. For aspirin and combination treatments, mice received aspirin (2.6mg/ml in drinking water) 2 days prior to tamoxifen administration and remained on aspirin till the end of the study. Animals were injected with BrdU (i.p.) 2 h prior to sampling tissues.

Immunohistochemistry (IHC)

Mouse intestines were flushed with water, cut open longitudinally, pinned out onto silicone plates and fixed in 10% neutral buffered formalin overnight at 4℃. Fixed tissue was rolled from the proximal to distal end into Swiss-rolls and processed for paraffin embedding. Tissue blocks were cut into 5μm sections and stained with haematoxylin and eosin (H&E). IHC was performed on formalin-fixed intestinal sections according to standard staining protocols. The primary antibody used was against BrdU (1:150, BD Biosciences, #347,580), and representative images are shown.

Statistical analysis

Data were presented and statistical analysis was performed using GraphPad Prism 9. Statistical tests were performed as stated, and significance was expressed as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Results are expressed as mean values with standard error of the mean (SEM) where independent experiments are compared and with standard deviation (SD) where technical replicates are compared. Here, technical replicates refer to separate wells or flasks of cells that are from the same original passage of cells, seeded and treated at the same time. Independent experiments refer to separate passages of cells that were seeded at different times.



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