Suppression of IFN-Induced Transcription Underlies IFN Defects Generated by Activated Ras/MEK in Human Cancer Cells

Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes

Activation of IFN-induced transcription in control SKOV3 and Ras-transformed SKOV3 cells.

<p>The expression of GBP2, IFIT2, MAP2, RIGI and STAT2 in control SKOV3 and Ras-transformed SKOV3 cells (clone 15) at 12 hours after IFN stimulation (0, 12.5, 50 and 100 U/ml) was determined by quantitative RT-PCR. The relative expression level was calculated compared to the untreated control SKOV3 cells after normalization against GAPDH expression levels. (n = 3, * P<0.05 and ** P<0.01 compared to IFN concentration-matched control).</p

Effect of U0126 treatment on the anti-viral IFN response in moderately resistant and completely resistant cell lines.

<p>(A) Cell lines were infected with VSV (MOI = 1) for 24 hours after treatment with IFN (0–5000 U/ml) with or without U0126 (0–20 µM) for 16 hours. Western blot analysis was used to detect viral protein (VSV-G) levels, the level of phosphorylated ERK (p-ERK) with GAPDH used as a loading control. The samples were analyzed on two membranes simultaneously using identical conditions for incubation and detection. One representative experiment out of 3 is shown. (B) Viral progeny production was determined after infection with VSV (MOI = 1) for 24 hours following treatment with IFN (50 or 2000 U/ml) and with U0126 (0, 5, 10 or 20 µM) for 16 hours.</p

Representative profiles of IFN sensitive, moderately resistant and completely resistant cell lines.

<p>IFN sensitive (A), moderately resistant (B) and completely resistant cell lines (C) were identified by pretreating cells with IFN (0, 10, 50, 100, 500, 1000 and 5000 U/ml) for 16 hours and then challenged with VSV at a MOI of 1 for 24 hours. Cell viability was determined using crystal violet staining and expressed as average percentage compared to the uninfected control wells (n = 3 wells). One representative experiment is shown.</p

Validation of changes in gene expression by quantitative RT-PCR.

<p>The level of gene expression in HT1080 cells left untreated, treated with IFN (50 U/ml), with U0126 (20µM) or with IFN and U0126 for 6 hours or 12 hours was determined by quantitative RT-PCR. The relative expression level was calculated compared to the 6 hour untreated control after normalization against GAPDH expression levels. (n = 3, * P<0.01 compared to time-matched control, ** P<0.05 compared to all other groups).</p

Microarray analysis of IFN inducible genes in IFN sensitive SKOV3 cells, moderately resistant HT1080 cells treated with IFN, U0126 or both.

<p>(A) Venn diagrams from DNA microarray analysis showing global suppression of IFN-regulated genes in HT1080 cells compared to SKOV3 cells. Shown are the number of genes significantly upregulated (FDR <0.01) at 6 hours after IFN treatment in the SKOV3 vs. HT1080 cell lines. (B) Venn diagrams showing the number of genes significantly upregulated (FDR <0.01) in HT1080 cells following treatment with IFN, U0126 or both IFN and U0126 for 6 hours or 12 hours as indicated. (C) Venn diagrams comparing genes upregulated by “protective” treatments in each cell type (IFN for SKOV3 cells and IFN/U0126 for HT1080 cells).</p

Gene ontology analysis of genes that are differentially expressed with combined IFN and U0126 treatment compared to the single treatments.

a<p>↑: increased representation, ↓: decreased representation, – no change.</p

Effect of Ras transformation on the efficiency of viral infection in IFN sensitive SKOV3 cells.

<p>(A) Western blot analysis using anti-Ras, phosphorylated ERK or GAPDH antibody to determine Ras/MEK activation in control SKOV3 and Ras transformed SKOV3 (clone 10 and 15). Control SKOV3 and Ras transformed SKOV3 cells were treated with IFN (0, 12.5, 25, 50 and 100 U/ml), and then challenged with challenged with VSV (MOI of 1). Infection was evaluated by (B) western blot analysis for VSV-G protein and GAPDH at 24 hours infection and by (C) progeny virus assay at 48 hours after infection.</p

Lifestyle, metabolite, and genetic determinants of formate concentrations in a cross-sectional study in young, healthy adults

BackgroundFormate is an important metabolite that serves as a donor of one-carbon groups to the intracellular tetrahydrofolate pool. However, little is known of its circulating concentrations or of their determinants.ObjectiveThis study aimed to define formate concentrations and their determinants in a healthy young population.DesignSerum formate was measured in 1701 participants from the Trinity Student Study. The participants were men and women, aged 18 to 28 y, enrolled at Trinity College, Dublin. Formate concentrations were compared with other one-carbon metabolites, vitamin status, potential formate precursors, genetic polymorphisms, and lifestyle factors.ResultsSerum formate concentrations ranged from 8.7 to 96.5 µM, with a mean of 25.9 µM. Formate concentrations were significantly higher in women than in men; oral contraceptive use did not further affect them. There was no effect of smoking or of alcohol ingestion, but the TT genotype of the methylenetetrahydrofolate reductase (MTHFR) 677C→T (rs1801133) polymorphism was associated with a significantly decreased formate concentration. Formate was positively associated with potential metabolic precursors (serine, methionine, tryptophan, choline) but not with glycine. Formate concentrations were positively related to serum folate and negatively related to serum vitamin B-12.ConclusionsFormate concentrations were sensitive to the concentrations of metabolic precursors. In view of the increased susceptibility of women with the TT genotype of MTHFR to give birth to infants with neural tube defects as well as the effectiveness of formate supplementation in decreasing the incidence of folate-resistant neural tube defects in susceptible mice, it will be important to understand how this genotype decreases the serum formate concentration. This trial was registered at www.clinicaltrials.gov as NCT03305900