Active RAS/MEK pathway downregulates expression of IFN-inducible genes by targeting IRF1: implications for understanding molecular mechanisms of viral oncolysis

Oncolytic viruses exploit common molecular changes in cancer cells, which are not present in normal cells, to target and kill cancer cells. Ras transformation and defects in type I interferon (IFN)-mediated antiviral responses are known to be the major mechanisms underlying viral oncolysis. The Hirasawa lab has previously demonstrated that oncogenic Ras/Mitogen-activated protein kinase kinase (Ras/MEK) activation suppresses the transcription of many IFN-inducible genes in human cancer cells, suggesting that Ras transformation underlies type I IFN defects in cancer cells. The objective of my PhD project was to elucidate the mechanisms underlying how Ras/MEK downregulates IFN-induced transcription. By conducting promoter deletion analysis of IFN-inducible genes, the IFN regulatory factor 1 (IRF1) binding site was identified to be responsible for the regulation of transcription by MEK. MEK inhibition promoted transcription of the IFN-inducible genes in wild-type mouse embryonic fibroblasts (MEFs), but not in IRF1− / − MEFs. Furthermore, IRF1 expression was lower in RasV12 cells compared with vector control NIH3T3 cells, which was restored to equivalent levels by inhibition of MEK. Similarly, MEK inhibition restored IRF1 expression in human cancer cells. IRF1 re-expression in human cancer cells increased cellular resistance to infection by the oncolytic vesicular stomatitis virus strain. Together, these results indicate that Ras/MEK activation in cancer cells downregulates transcription of IFN-inducible genes by targeting IRF1 expression, resulting in increased susceptibility to viral oncolysis. I further sought to determine how active Ras/MEK downregulates IRF1 expression. MEK inhibition restored IRF1 expression at the protein level prior to mRNA induction; however, it did not affect IRF1 protein stability. The expression of IRF1- targeting microRNA, activity of IRF1 5’ and 3’-UTRs, and polysome loading of IRF1 mRNA in response to MEK inhibition were analyzed; however, the translational regulation of IRF1 mRNA by Ras/MEK remained inconclusive. To determine whether Ras/MEK modulates post-translational modifications (PTMs) of IRF1, phosphorylation, ubiquitination, sumoylation, and acetylation of IRF1 were examined. MEK inhibition promoted ubiquitination and inhibited sumoylation of IRF1, indicating that active Ras/MEK alters PTM of IRF1 protein. Lastly, siRNA screens and overexpression experiments identified RSK3 and RSK4 to be the ERK downstream effectors involved in Ras/MEK-mediated IRF1 regulation

視覚野においてT型Ca²⁺チャネル依存性長期増強を引き起こすためにはTNFαが必要である

Monocular deprivation produces depression and potentiation of visual responses evoked in visual cortical neurons by stimulation of deprived and nondeprived eyes, respectively, during the critical period of ocular dominance plasticity. Our previous studies suggested that T-type Ca²⁺ channel-dependent long-term potentiation (LTP), induced by 2 Hz stimulation, mediates the potentiation of visual responses. However, it was proposed that the experience-dependent response potentiation is mediated by tumor necrosis factor-α (TNFα)-dependent homeostatic synaptic scaling but not by Hebbian synaptic plasticity, because the potentiation was absent in TNFα knockout (TNFα-KO) mice. In this study, we investigated whether TNFα is required for LTP induced by 2 Hz stimulation using visual cortical slices prepared from critical period mice and rats. The production of LTP was prevented by pharmacological blockade of TNFα in rats and mice. LTP production was also prevented by an inhibitor of TNFα-converting enzyme that converts membrane-bound TNFα to soluble TNFα. In TNFα-KO mice, LTP did not occur and was rescued by exogenous soluble TNFα. Soluble TNFα was required for LTP production only during a restricted time window soon after 2 Hz stimulation. These results strengthen the view that T-type Ca²⁺ channel-dependent LTP contributes to the potentiation of nondeprived eye responses following monocular deprivation.博士（医学）・乙第1401号・平成29年6月28日Copyright © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis

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