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Dichotomal functions of phosphorylated and unphosphorylated STAT1 in hepatocellular carcinoma.

ABSTRACT: Interferons (IFNs) with antiviral and immune-stimulatory functions have been widely used in prevention and treatment of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 1 (STAT1) is a key element of the IFN signaling, and the function of STAT1 is critically determined by its phosphorylation state. This study aims to understand the functions of phosphorylated (p-) and unphosphorylated (u-) STAT1 in HCC. We found that u-STAT1 is significantly elevated in patient HCC tumor tissues and predominantly expressed in cytoplasm; while p-STAT1 is absent. Loss of u-STAT1 potently arrested cell cycle and inhibited cell growth in HCC cells. Induction of p-STAT1 by IFN-α treatment effectively triggers the expression of interferon-stimulated genes (ISGs), but has moderate effect on HCC cell growth. Interestingly, both u-STAT1 and p-STAT1 are induced by IFN-α, through with distinct time-dependent process. Furthermore, the ISG induction patterns mediated by p-STAT1 and u-STAT1 are also distinct. Importantly, artificial blocking of the induction of u-STAT1, but not p-STAT1, sensitizes HCC cells to treatment of IFNs. Therefore, p-STAT1 and u-STAT1 exert dichotomal functions and coordinately regulate the responsiveness to IFN treatment in HCC. KEY MESSAGES: STAT1 is upregulated and predominantly presented as u-STAT1 in HCC, while p-STAT1 is absent. U-STAT1 sustains but p-STAT1 inhibits HCC growth. The dynamic change of phosphorylation state of STAT1 control the responsiveness to IFN treatment.

SUBMITTER: Ma B

PROVIDER: S-EPMC6326978 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundUnphosphorylated signal transducer and activator of transcription 1 (U-STAT1) has been reported to elicit a distinct gene expression profile as compared to tyrosine-phosphorylated STAT1 (P-STAT1) homodimers. However, the impact of U-STAT1 on the IFNγ-induced immune response mediated by P-STAT1 is unknown. By generating a double mutant of STAT1 with mutation R602L in the Src-homology 2 (SH2) domain and Y701F in the carboxy-terminal transactivation domain mimicking U-STAT1, we investigated the effects of U-STAT1 on P-STAT1-mediated signal transduction.ResultsIn this study, we discovered a novel activity of U-STAT1 that alters the nucleo-cytoplasmic distribution of cytokine-stimulated P-STAT1. While the dimerization-deficient mutant R602L/Y701F was not able to display cytokine-induced nuclear accumulation, it inhibited the nuclear accumulation of co-expressed IFNγ-stimulated wild-type P-STAT1. Disruption of the anti-parallel dimer interface in the R602L/Y701F mutant via additional R274W and T385A mutations did not rescue the impaired nuclear accumulation of co-expressed P-STAT1. The mutant U-STAT1 affected neither the binding of co-expressed P-STAT1 to gamma-activated sites in vitro, nor the transcription of reporter constructs and the activation of STAT1 target genes. However, the nuclear accumulation of P-STAT1 was diminished in the presence of mutant U-STAT1, which was not restored by mutations reducing the DNA affinity of mutant U-STAT1. Whereas single mutations in the amino-terminus of dimerization-deficient U-STAT1 similarly inhibited the nuclear accumulation of co-expressed P-STAT1, a complete deletion of the amino-terminus restored cytokine-stimulated nuclear accumulation of P-STAT1. Likewise, the disruption of a dimer-specific nuclear localization signal also rescued the U-STAT1-mediated inhibition of P-STAT1 nuclear accumulation.ConclusionOur data demonstrate a novel role of U-STAT1 in affecting nuclear accumulation of P-STAT1, such that a high intracellular concentration of U-STAT1 inhibits the detection of nuclear P-STAT1 in immunofluorescence assays. These observations hint at a possible physiological function of U-STAT1 in buffering the nuclear import of P-STAT1, while preserving IFNγ-induced gene expression. Based on these results, we propose a model of a hypothetical import structure, the assembly of which is impaired under high concentrations of U-STAT1. This mechanism maintains high levels of cytoplasmic STAT1, while simultaneously retaining signal transduction by IFNγ. Video Abstract.

Project description:ObjectivesTo reveal the mechanisms of the effects of mortalin in hepatocellular carcinoma (HCC) and to identify potential novel chemical inhibitors of mortalin.Materials and methodsFor the experiments, three HCC cell lines (HepG2 cells, Hep3B cells, and sorafenib-resistant HuH7 cells) and xenografted nude mice were used. For the clinical analysis, cohorts of 126 patients with HCC and 34 patients with advanced recurrent HCC receiving sorafenib therapy were examined.ResultsMortalin regulated the phosphorylation-modification of cancer-associated proteins and also regulated angiogenesis-related secretome to cause angiogenesis and sorafenib resistance in HCC cells. Two molecular mechanisms were identified. In one, via phosphatidylinositol 3-kinase (PI3K)/Akt signaling, mortalin regulated nuclear factor (NF)-κB and then activated vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR)2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to neovascularization. In the other, mortalin regulated PI3K/Akt/β-catenin and then regulated Bcl-XL and Bcl-2, leading to the antiapoptosis effect of HCC. Treatment of the sorafenib-resistant xenografts with sorafenib in combination with mortalin knockdown facilitated the sorafenib-mediated inhibition of tumor growth and angiogenesis and increased apoptosis. Mortalin was a potential risk factor for HCC, predicting poor prognosis and sorafenib resistance. Finally, we showed that caffeic acid (C9H8O4) could bind to and induce the ubiquitination-mediated degradation of mortalin, which in turn blocked the abovementioned signaling pathways, leading to the inhibition of angiogenesis and the reversal of sorafenib resistance.ConclusionsMortalin, which regulates the phosphorylation of cancer-associated proteins, caused angiogenesis and sorafenib resistance, and was a competitive risk factor for HCC. Caffeic acid can therefore be considered a novel chemical inhibitor that targets the action of mortalin and a potential treatment for HCC.

Project description:Interferon-gamma stimulation triggers tyrosine phosphorylation of the transcription factor STAT1 at position 701, which is associated with switching from carrier-independent nucleocytoplasmic shuttling to carrier-mediated nuclear import. Unlike most substrates that carry a classical nuclear localization signal (NLS) and bind to importin alpha1, STAT1 possesses a nonclassical NLS recognized by the isoform importin alpha5. In the present study, we have analyzed the mechanisms by which importin alpha5 binds phosphorylated STAT1 (pSTAT1). We found that a homodimer of pSTAT1 is recognized by one equivalent of importin alpha5 with K(d)=191+/-20 nM. Whereas tyrosine phosphorylation at position 701 is essential to assemble a pSTAT1-importin alpha5 complex, the phosphate moiety is not a direct binding determinant for importin alpha5. In contrast to classical NLS substrates, pSTAT1 binding to importin alpha5 is not displaced by the N-terminal importin beta binding domain and requires the importin alpha5 C-terminal acidic tail (505-EEDD-508). A local unfolding of importin alpha5 Armadillo (ARM) repeat 10 accompanies high-affinity binding to pSTAT1. This unfolding is mediated by a single conserved tyrosine at position 476 of importin alpha5, which is inserted between ARM repeat 10 helices H1-H2-H3, thereby preventing intramolecular helical stacking essential to stabilize the folding conformation of ARM 10. Introducing a glycine at this position, as in importin alpha1, disrupts high-affinity binding to pSTAT1, suggesting that pSTAT1 recognition is dependent on the intrinsic flexibility of ARM 10. Using the quantitative stoichiometry and binding data presented in this article, together with mutational information available in the literature, we propose that importin alpha5 binds between two STAT1 monomers, with two major binding determinants in the SH2 and DNA binding domains. In vitro, this model is supported by the observation that a 38-mer DNA oligonucleotide containing two tandem cfosM67 promoters can displace importin alpha5 from pSTAT1, suggesting a possible role for DNA in releasing activated STAT1 in the cell nucleus.

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Dichotomal functions of phosphorylated and unphosphorylated STAT1 in hepatocellular carcinoma.

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