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

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

Coordinate downregulation of IFN-γ inducible HLA-II expression by E₂ is reversed by ICI-mediated degradation of ERα in MC2 cells.

<p>VC5 and MC2 cells were cultured in E₂-depleted media, treated with vehicle (ethanol), E₂ (10⁻⁹ M) or/and ICI (10⁻⁶ M) followed by stimulation with IFN-γ (100 U/ml) for 96 hours. HLA-II expression was analyzed by surface flow cytometry using (A) anti-DR, (L243), and intracellular flow cytometry using (B) anti-DM (Map.DM1) and (C) anti-Ii (LN2). Bar graphs represent the MFI ± SEM of three independent experiments. (*p<0.05, **p<0.01). (D) Western blot analysis was performed on whole cell extracts using for HLA-DRα (TAL 1B5), HLA-DM (TAL18.1) and Ii (LN2); GAPDH (Ab8245) is the protein loading control. Bar graphs show the ratio of band intensities, normalized to GAPDH band intensities and represent the mean ± SEM ratio of three independent experiments: (E) HLA-DRα/GAPDH (F) HLA-DM/GAPDH, and (G) Ii/GAPDH (* p<0.05, ** p<0.01).</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

E₂-ERα signaling down regulates CIITA protein and mRNA expression in ER⁺ BCCL.

<p>VC5 and MC2 cells were cultured in E₂-depleted media, treated with vehicle (ethanol), E₂ (10⁻⁹ M) or/and ICI (10⁻⁶ M) and stimulated or not with IFN-γ (100 U/ml) for 24 and 4 hours, for CIITA protein and mRNA expression, respectively. (A) Western blot analysis was performed on cytoplasmic and nuclear extracts for CIITA (antiserum #21) and ERα (HC-20). (B) Cytoplasmic CIITA and nuclear CIITA were normalized to GAPDH and P84 respectively; bar graphs represent the mean ± SEM ratio of three independent experiments (**p<0.01). (C) CIITA mRNA was relatively quantified by real time PCR using Taqman gene expression assay. GAPDH was used as an endogenous control and the data were expressed relative to a control B cell line (RAJI). Bar graphs represent the mean ± SEM of three replicate assays (**p<0.01).</p

E₂-ERα signaling pathway interferes with CIITA pIV activity in MC2.

<p>VC5 and MC2 cells were cultured in E₂-depleted media followed by transfection with CIITA pIV luciferase constructs. On the following day, cells were treated with vehicle (ethanol), E₂ (10⁻⁹ M) and/or ICI (10⁻⁶ M), and stimulated or not with IFN-γ (100 U/ml) for 12 hours. Data are expressed as fold induction over the PGL2 Basic empty plasmid after controlling for transfection efficiency using cells dual transfected with GFP (Green Florescent Protein). The effect of ERα on the transcription activation of CIITA PIV was determined from relative luciferase activities in transfected MC2. Error bars represent the mean ± SEM of three independent experiments (**p<0.01).</p

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

Mutation of putative ERE sites in CIITA pIV does not enhance CIITA pIV activation in MC2.

<p>(A) CIITA pIV nucleotide sequence from −346 to +50 with the GAS and IRF1 binding sites (shaded hexagon) and the predicted ERE (clear rectangles) were identified using online transcription factor prediction software, (<a href="http://tfbind.hgc.jp/" target="_blank">http://tfbind.hgc.jp/</a>, <a href="http://alggen.lsi.upc.es/" target="_blank">http://alggen.lsi.upc.es/</a> and <a href="http://www.cbrc.jp/index.eng.html" target="_blank">http://www.cbrc.jp/index.eng.html</a>). Site directed mutagenesis was used to perform deletion of the predicted ERE. (B) VC5 and MC2 were transfected with CIITA pIV constructs, then treated with vehicle (ethanol) or E₂ (10⁻⁹ M) and stimulated with IFN-γ (100 U/ml) for 12 hours, followed by determination of luciferase activity. Bar graphs represent the mean ± SEM of three independent experiments (**p<0.01, ***p<0.001).</p