Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1–/–). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1–/– and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/– and hTS/Men1–/– mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

Islet hyperplasia was scored when multiple islets were greater than 100 microns and had normal architecture. For islet cell adenoma, architecture was similar to hyperplasia, but size of islet was larger with compression of adjacent acinar pancreas and more prominent vascular dilation. Islet cell carcinoma showed capsular invasion and increased cellular pleomorphism, fibrosis and mitotic figures.

Establishment of stable clones of MEF cells with hTS overexpression
MEF wild type (clone W10) and MEF-Men1-null (clone N41.4) cell lines were obtained from Dr. Chandresekharappa (NIH) (5) and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37°C under 5% CO2. To generate stable cell lines overexpressing TS, TS cDNA was cloned into a retroviral expression vector, pLNCX (Clontech) resulting in pLNCX-TS. To construct the pLNCX-TS expression vector, a 1965 bp fragment of TS cDNA plus 3'UTR was excised from pOTB7-TS using ScaI and SmaI sites and the fragment was subcloned into HpaI site in pLNCX vector. To generate retrovirus, HEK293T cells were transfected with 10 μg of pLNCX-TS or pLNCX empty vector, and 2.5 μg retroviral packaging plasmids (pMD-MuLV and pMD-G, Addgene) using Effectene (Qiagen Cat# 301427) as per the manufacturer's instructions. Briefly, the transfection mixture was prepared by adding pLNCX-TS or pLNCX plasmid, the enhancer reagent provided in the Qiagen kit (16 µl) and Effectene (60 μI) to EC buffer (300 µl) and incubated for 10 min, followed by addition of 3 ml of 10% DMEM to the mix. HEK293T cells were seeded in a T75 flask at a density of 2 x 10 6 cells and the retroviral mixture was added 24h after plating. Transfection mixture was removed from the flask after 8h, replaced with fresh medium and cells were incubated for an additional 48h. Media was then collected and spun at 3000 g for 3 min to remove residual HEK293T cells and the supernatant was transferred to a new tube and filtered through a 0.45 μm syringe filter.
The virus preparation was kept at -80°C until use.
For transduction, 1 x 10 6 MEF wild type and MEF-Men1 -/cells were seeded in 100-mm dishes, then media was removed after 24h and plates were incubated with 4 ml of viral supernatant (containing 2 μg /ml of polybrene (Millipore) for 24h to transduce cells with retroviral TS cDNA or vector control (pLNCX-TS and pLNCX) (6). After 24h, virus containing media was removed and replaced with selection media (DMEM supplemented with 10% FBS, 1% Penicillin-Streptomycin and G418 [300 µg/ml]). Cells were incubated with selection media for 2 weeks, with media change every two days, to select single cell clones that stably overexpress TS or control vector (designated as MEF-V, MEF-V/Men1 -/-, MEF-TS and MEF-TS/Men1 -/-). Selected single cell clones for each assay are described in detail below. After expansion and harvest, TS expression was determined by immunoblot analysis.

Cell viability assay
Viability of cells was measured using MTS reagent as recommended by manufacturer instructions (Promega). Distinct clones of MEF-TS, MEF-TS/Men1 -/cells and their corresponding control vector expressing cells were seeded in 96-well plates (1 x 10 3 cells/well; each clone was seeded into six wells in a 96 well plate). Plates were incubated for 24, 48 and 72 hours. At each time point, 10 μI of MTS reagent was added to each well without removing the media, incubated at 37°C for 1 hour and assayed using a micro plate reader at 490 nm wavelength (SpectraMax M3, Molecular Devices).

Cell cycle analysis
Cell cycle analysis was performed by flow cytometry (FACSort; Becton Dickinson). Cells (1 x 10 6 ) were harvested, washed 2x with PBS and suspended in 200 ul of PBS. Cells were fixed using 4 ml of cold 75% ethanol at 4°C overnight and then washed 2x with PBS.

Immunohistochemistry of human tissue
Immunohistochemical analysis of TS, ɣ-H2AX and Ki-67 in human patient samples was performed using three tissue array blocks containing 88 human pancreatic neuroendocrine tumors prepared as described previously (7). Briefly, core tissue biopsies (2 mm in diameter) were taken from individual paraffin-embedded tumors (donor blocks) and arranged in recipient paraffin blocks (tissue array blocks) using a trephine. A representative core was sampled in each tumor tissue and an adequate case was defined as a tumor occupying more than 10% of the core area. Four µm sections were cut from each tissue array block, deparaffinized and dehydrated. Immunohistochemical staining for TS (1:100, Fisher Scientific Cat# MAB4130MI) ɣ-H2AX (1:300, Upstate Cat# 05-636) and Ki-67 (1:500, Abcam Cat# ab92742) was performed using an automated immunostainer (Ventana BenchMark XT, Ventana Medical System, Tucson AZ, USA).
Staining intensity and stained tumor cell percentage of TS were measured as previously described (7). Staining intensity was classified as negative, weak and strong expression.
For statistical analyses, the staining was considered as positive when present in more than 5% of the tumor cell population. For ɣ-H2AX and Ki-67 immunohistochemistry stained slides were scanned using an Aperio AT2 scanner (Leica Biosystem, Newcastle upon Tyne, UK), then percentage of ɣ-H2AX and Ki-67 positive cells were evaluated using QuPath (open-source software) (8).

Comet Assay
MEF cells were cultured for 48 h before being collected using trypsin and resuspended in low-melting agarose (R&D Systems) at a final concentration of 0.9% before spreading on 20-well Comet Slides (R&D Systems). Comet slides were incubated in lysis buffer (R&D Systems) for 1 h, at 4°C to lyse cells before washing twice with deionized H2O and submerging Comet Slides in an alkaline electrophoresis solution (deionized H2O containing 200 mM NaOH and 1 mM EDTA, pH > 13) for 20 minutes at room temperature.
Electrophoresis was run for 30 min with applied voltage of 21 V and a current of approximately 300 mA using the Comet Assay Electrophoresis System II (R&D Systems).
The electrophoresis unit and buffer were chilled to 4°C. Slides were rinsed twice in deionized H2O, fixed in ethanol for 5 minutes, and dried for 10 minutes at 37°C using a standard incubator. DNA was stained with 40 µl 1x SYBR Gold nucleic acid gel stain (S-11494, Life Technologies) per well and immediately visualized using the Leica DM6000B upright microscope. DNA damage was quantified using the OpenComet Image-J plugin

Immunoblot analysis
Tissues were homogenized using the Omni general laboratory homogenizer (GLH) following the manufacturer's protocol (OMNI international). Cell lines were collected by scraping and washed 2x with cold PBS. Cells and tissue homogenates were lysed in a RIPA lysis buffer (Santa-Cruz, sc-24948) and centrifuged (12000 rpm, 20 min) to obtain whole-cell lysates. Protein concentration was measured using the Bradford assay.
Aliquots of the lysates (10-20 μg of protein) were separated on 10% or 4-20% Tris-glycine gels, transferred to nitrocellulose membranes, and probed with specific antibodies (Supplemental Table 9); Blots were visualized by Super Signal Chemiluminescent Dura substrate (ThermoFisher Scientific). For densitometric analysis of proteins, Image-J software was used for each immunoblot to quantify band intensity, and then each band intensity value was normalized to loading control.

Big Blue® mutation detection assay
Men1 -/-/BB and hTS/Men1 -/-/BB mice carry bacteriophage lambda shuttle vector (LIZ) containing CII gene as the target of mutagenesis. The mutation detection assay was carried out using the Transpak ® and the λ Select-cII TM protocol (Agilent Technology).
Genomic DNA was isolated from mice pancreas at 5 months and 10 months of age.
Genomic DNA was packaged in vitro to produce virulent lambda phage, infecting E. coli strain G1250 and plated. Plates were incubated under mutant selective (24°C, 40 h) or control (37°C, 24 h) conditions. Approximately 300,000 ~ 800,000 plaque forming units (PFUs) were analyzed from multiple sample preparations of pancreas tissue or pancreas tumors of age matched groups of mice. The mutation frequency was calculated as the ratio of the number of mutant plaques (grown at 24°C, 40 h) per total number of plaques (grow at 37°C, 24 h). Normalization of mutation frequency was calculated by subtracting the false positive plaques derived from 5 months old Men1 -/-/BB mice. 195 mutant plaques were isolated using sterile pipette tips, PCR amplified and sequenced to further verify the type of mutations present in the sequence. Primers for PCR amplification and sequencing of CII gene are cll-F 5´-CCACACCTATGGTGTATG-3´ and cll-R 5´-CCTCTGCCGAAGTTGAGTAT-3´.

Metaphase preparation and spectral Karyotype analysis (SKY)
For chromosome aberration analysis, MEF-V/Men1 -/and MEF-TS/Men1 -/cells (0.5 x 10 6 cells) were seeded in a 100 mm dish, and incubated for 48h. Cells were blocked in metaphase by treatment with 0.01 mg/ml colcemid in HBSS (Karyomax, Invitrogen) at 37°C for 3 h and were further harvested by trypsinization, and pelleted by centrifugation (1000 rpm for 10 min). Cell pellets were suspended in 3 ml of hypotonic solution (0.075 M potassium chloride) and incubated at 37°C for 10 min. The cells were pelleted further by centrifugation and resuspended in 5 ml of 3:1 methanol/acetic acid for fixation.
Metaphase slides were prepared by dropping 20 μl of fixed cell suspension onto a clean slide and incubated at 42°C overnight. Metaphases were hybridized with the 20-color mouse SKY paint kit according to manufacturer's protocol (Applied Spectral Imaging Inc).
Spectral images of the hybridized metaphases were acquired using a SD301 SpectraCubeTM system (Applied Spectral Imaging Inc) mounted on top of an epifluorescence microscope Axioplan 2 (Zeiss). Images were analyzed using Spectral Imaging 4.0 acquisition software (Applied Spectral Imaging Inc). G banding was simulated by electronic inversion of DAPI-counterstaining. Spectral karyotype analysis procedure was performed at the Comparative Molecular Cytogenetics Core Facility at NCI-Frederick.

Immunofluorescence analysis for γH2AX staining
MEF-TS (TS1-1), MEF-TS/Men1 -/cells (TS2-1) and their corresponding vector control cells (clones V1-1 and V2-1, respectively) were plated in a coverslip placed in 6 well plate (1 x 10 6 cells per well) and incubated for 24h. Cells were fixed in 4% paraformaldehyde for 10 min. After fixation, cells were permeabilized with 0.5% Triton X100 diluted in PBS and then blocked with 10% FBS in phosphate saline buffer (PBS) for 30 min. Cells were incubated with primary antibodies against γH2AX for 1 h at room temperature and with Alexa Fluor-conjugated secondary antibodies. The cells were mounted using VectaShield hard-set mounting media containing DAPI (Vector laboratories). Fluorescent images were recorded using an Olympus DP70 digital camera coupled to an Olympus IX71 inverted microscope (Olympus Corp). Percent fluorescence expression efficiency was measured using Image-J software.

Lentivirus production and lentivirus-mediated TS inhibition in BON cells
To produce lentiviral TS shRNA for transduction, HEK293T cells (12 x 10 6 ) were seeded in T175 flask and incubated overnight at 37ºC and 5% CO2 for 10-20h.

Immunohistochemistry of chromogranin A and synaptophysin
Immunostaining was performed in paraffin-embedded tissues. In brief, 4 µm sections were de-paraffinized, then treated with Citra Steam (Biogenex) for 30 minutes.

Spectrometry (UHPLC-HRMS)-based metabolomics data collection and analysis
MEF-V (clone V1-1), MEF-TS (clone TS1-1), MEF-V/Men1 -/-(clone V2-1) and MEF-TS/Men1 -/-(clone TS2-5) were used for the metabolomics data analysis. UHPLC-HRMSbased untargeted metabolomics was used for data collection. Chromatographic separation for metabolomics was achieved using reversed-phase chromatography with a C18-pfp column (Ace, Aberdeen, Scotland; 100 x 2.1 mm, 2 µm). The mobile phases consisted of solvent A (0.1% formic acid in H2O) and solvent B (acetonitrile). The system was held constant from 0-3 min at 100% A, then mobile phase B was ramped from 0% B to 80% over 10.0 min (3-13 min) and then held constant at 80% B for 3 min (13-16 min) with a flow rate of 350 µL/min and column temperature of 25°C. For equilibration, the system was returned to initial conditions with 0% B and the flow rate was increased to 600 µL/min. The flow rate was reduced back to 350 µL/min before the next injection. The data collection time per sample was 20.5 min. Both positive (injection volume 2 µL) and negative ion polarity (injection volume 3 µL) in full scan mode (35,000 mass resolution) were acquired.
For metabolomics data analysis, metabolite identification was performed with MZmine 2.0 and matching metabolite retention time and m/z value to an internal library of over 1000 metabolites representing level 1 identification following metabolomics standards initiative guidelines. Metabolic pathway analysis was conducted as previously mentioned (10).
Briefly, metabolic pathway analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database by matching metabolite sets with human metabolome (https://www.genome.jp/kegg/pathway.html). Metabolite set enrichment (fold enrichment) was further investigated using MetaboAnalyst (open-source R package). Pathway impact score was computed from pathway topological analysis using relative-betweenness centrality.

Secondary analysis of data sets from patient tumor samples
PanNET transcriptomic patient data was extracted from GSE117853 (subseries: GSE117851). List of genes related to DNA damage response was obtained from https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repairgenes.html. Correlation between TS expression and other genes related to nucleotide synthesis as well as DNA damage was analyzed in Graph Pad Prism. Pearson correlation between -1 and 1, with p value ≤ 0.05 was considered statistically significant. Data to study TS gene expression in patient samples with mutations in ATRX, DAXX, or MEN1 genes was obtained from GSE117853 (subseries: GSE117851).
To study the correlation between TS expression and mutation rate in patient samples of different tumor origin, we determined TS expression in human patients from 6 tumor types included in The Cancer Genome Atlas (11)