STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.Lukas Kenner and Jan Pencik are supported by FWF, P26011 and the Genome Research-Austria project “Inflammobiota” grants. Helmut Dolznig is supported by the Herzfelder Family Foundation and the Niederösterr. Forschungs-und Bildungsges.m.b.H (nfb). Richard Moriggl is supported by grant SFB-F2807 and SFB-F4707 from the Austrian Science Fund (FWF), Ali Moazzami is supported by Infrastructure for biosciences-Strategic fund, SciLifeLab and Formas, Zoran Culig is supported by FWF, P24428, Athena Chalaris and Stefan Rose-John are supported by the Deutsche Forschungsgemeinschaft (Grant SFB 877, Project A1and the Cluster of Excellence --“Inflammation at Interfaces”). Work of the Aberger lab was supported by the Austrian Science Fund FWF (Projects P25629 and W1213), the European FP7 Marie-Curie Initial Training Network HEALING and the priority program Biosciences and Health of the Paris-Lodron University of Salzburg. Valeria Poli is supported by the Italian Association for Cancer Research (AIRC, No IG13009). Richard Kennedy and Steven Walker are supported by the McClay Foundation and the Movember Centre of Excellence (PC-UK and Movember). Gerda Egger is supported by FWF, P27616. Tim Malcolm and Suzanne Turner are supported by Leukaemia and Lymphoma Research.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms873

PKR Transduces MDA5-Dependent Signals for Type I IFN Induction.

Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVΔE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluΔNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2α, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVΔE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism

PKR is required for IRF3 activation during VVΔE3L infection.

<p>WT and <i>Pkr</i>^<i>-/-</i> MEFs were infected with the indicated viruses (VVΔE3L and EMCV: MOI = 3, SeV: 200 HA units/mL) for 8 h. IRF3 nuclear translocation and total IRF3 expression were measured by western blotting of nuclear and cytoplasmic extracts. Detection of histone H3 and tubulin were used as loading controls for nuclear and cytoplasmic compartments, respectively. The data shown are representative of three independent experiments.</p

Interaction between MDA5 and PKR.

<p>(A) HEK293T cells were transfected with the indicated flag-tagged plasmids, infected with VVΔE3L (MOI = 3) for 8 h, and analyzed by immunoprecipitation and immunoblotting, as indicated. (B) Extracts from HEK293T cells transfected with flag-tagged MDA5 were untreated or treated with micrococcal nuclease (MNase) prior to flag immunoprecipitation and immunoblotting. (C) Extracts from A549 cells untreated or treated with IFN-α (100 u/ml for 8 h) were analyzed by immunoprecipitation and immunoblotting, as indicated. Input levels represent 10% of the cell lysates used for immunoprecipitation. (D) A549 cells expressing shNS or shPKR constructs were transduced with lentiviruses expressing constitutively active truncated RIG-I (RIG-I-CARD) or full-length MDA5, and analyzed for IFNβ expression 48 h post transduction by qRT-PCR. (E) IFNβ luciferase activity of HEK293T cells transiently expressing RIG-I-CARD, constitutively active full length RIG-I (RIGI-MIII), and MDA5, along with IFNβ-luc and βGal reporter plasmids. Cells were analyzed 48 h post transfection. (F) IFNβ luciferase activity was measured as in (E) for HEK293T cells transfected with constructs expressing the CARD domains of either RIG-I (RIG-I-CARD) or MDA5 (MDA5-CARD). shNS = short-hairpin against non-specific RNA; shPKR = short-hairpin against PKR mRNA; RLU = relative luciferase units. Significant differences (p< 0.01) are indicated (*).</p

Ectopic PKR activation induces IFNβ expression in the absence of viral infection.

<p>(A) HT1080-GyrB-PKR cells were treated with 100 ng/ml coumermycin (coum) for 8 or 24 h, followed by western blot analysis for phospho-eIF2α. (B, C) HT1080-GyrB-PKR and HT1080-GyrB-PKR-K296H cells were treated with coumermycin or IFNγ for 8 h followed by qRT-PCR analysis to quantify expression of IFNβ (B) or GBP1 (C). Significant difference (p< 0.001) is indicated (*). (D) HT1080-GyrB-PKR cells were treated either with coumermycin (8 h) or infected with VVΔE3L (6 h), then assayed for IRF3 phosphorylation by western blotting. Detection of total IRF3 and HDAC3 indicated equal protein recovery.</p

PKR-induced IFNβ expression requires MAVS.

<p>HT1080-GyrB-PKR cells expressing scrambled or shRNAs specific for MDA5 (A, B, C) or MAVS (D, E, F) were treated with coumermycin, IFNγ, or infected with VVΔE3L or VSV (MOI = 3) for 8 h, and scored by qRT-PCR either for MDA5, MAVS, IFNβ or GBP1 mRNA expression.</p

PKR is required for VVΔE3L-mediated IFNβ induction.

<p>(A) Infection of WT or <i>Pkr</i>^<i>-/-</i> MEFs with VVΔE3L (MOI = 3) or FluΔNS1 (MOI = 1) for 8 h followed by quantifying IFNβ expression by qRT-PCR. (B) Treatment of WT and <i>Pkr</i>^<i>-/-</i> MEFs with polyriboinosinic:polyribocytidylic acid (pIC) for 4 h followed by analysis of IFNβ expression by qRT-PCR. (C) A549 shNS (nonspecific) and A549 shPKR cells were infected with VVΔE3L or FluΔNS1 for 8 h and assayed for IFNβ mRNA by qRT-PCR. Data represents the mean and standard deviation from triplicate samples. Significance (*) was determined by an unpaired t-test (p< 0.001).</p

MDA5 enhances VVΔE3L-induced PKR catalytic activity.

<p>(A) <i>Mda5</i>^<i>+/-</i> and <i>Mda5</i>^<i>-/-</i> MEFs were infected with VVΔE3L and cell lysates were harvested at 8 h post infection. PKR immunoprecipitates were assessed for catalytic activity by <i>in vitro</i> kinase assay, and levels of phosphorylated PKR were quantified by phosphorimager (upper panel). Total PKR and VV I3L protein levels (lower panel) were detected by western blotting. (B) Extracts from cells infected with VVΔE3L or SeV were analyzed by <i>in vitro</i> kinase assay as in (A). Antibodies to I3L and VSV-G were used to document equal levels of virus infection in WT and KO cells.</p

VVΔE3L signals through IRF3 and MDA5, but not RIG-I.

<p>(A) Wild type (WT) and <i>Irf3</i>^<i>-/-</i> mouse embryonic fibroblasts (MEFs) were infected with vaccinia virus lacking E3L (VVΔE3L) (MOI = 3) for 8 h and IFNβ expression was measured by quantitative real time PCR (qRT-PCR). (B) Infection of <i>RigI</i>^<i>-/-</i> and <i>Mda5</i>^<i>-/-</i> MEFs and corresponding WT counterparts with VVΔE3L (MOI = 3) for 8 h. IFNβ expression was quantified by qRT-PCR. (C) Same as in (B) for encephalomyocarditis virus infection (EMCV) (MOI = 3). (D) Same as in (B) following Sendai virus (SeV) (100 HA units/mL) infection. Data shown represent means and standard deviations of three independent experiments. Statistically significant differences (p< 0.001) are indicated (*).</p