A single high dose of interferon-β (IFNβ) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNβ-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNβ, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNβ-induced proteins, and to constitutive resistance to DNA damage.

In normal human cells treated with interferons (IFNs), the concentration of tyrosine-phosphorylated STAT1 (YP-STAT1), which drives the expression of a large number of genes, increases quickly but then decreases over a period of several hours. Because the STAT1 gene is activated by YP-STAT1, IFNs stimulate a large increase in the concentration of unphosphorylated STAT1 (U-STAT1) that persists for several days. To test the significance of high U-STAT1 expression, we increased its concentration exogenously in the absence of IFN treatment. In response, the expression of many immune regulatory genes (e.g., IFI27, IFI44, OAS, and BST2) was increased. In human fibroblasts or mammary epithelial cells treated with low concentrations of IFN-␤ or IFN-␥, the expression of the same genes increased after 6 h and continued to increase after 48 or 72 h, long after the concentration of YP-STAT1 had returned to basal levels. Consistent with its activity as a transcription factor, most U-STAT1 was present in the nuclei of these cells before IFN treatment, and the fraction in nuclei increased 48 h after treatment with IFN. We conclude that the nuclear U-STAT1 that accumulates in response to IFNs maintains or increases the expression of a subset of IFN-induced genes independently of YP-STAT1, and that many of the induced proteins are involved in immune regulation. Their signaling pathways are mediated by the sequential phosphorylation of Janus family kinases (JAKs) and STATs. Type I IFNs phosphorylate STATs 1 and 2, which form IFN-stimulated gene factor-3 (ISGF-3), a ternary complex that also includes IFN response factor-9 (IRF9). IFN-␥ induces the phosphorylation of STAT1, which forms STAT1 homodimers. These activated transcription factors translocate into the nucleus, where they bind to distinct conserved sequences in the promoters of target genes. However, recent studies have shown that IFN signaling is much more complex (2). IFNs activate several different kinases in addition to the JAKs, other STATs in addition to STAT1 and STAT2, and even other transcription factors (3-9).Our previous work has shown that STAT1 drives the constitutive expression of some genes without phosphorylation (10, 11). For example, the complex of unphosphorylated STAT1 (U-STAT1) and IRF1 mediates the constitutive expression of the low-molecular mass polypeptide 2 (LMP2) gene (11). Similarly, Cui et al. (12) have shown that unphosphorylated STAT6 cooperates with p300 to increase transcription of the cyclooxygenase-2 gene. Initially, unphosphorylated STATs were considered to be latent transcription factors in the cytoplasm, entering the nucleus to induce gene expression only in response to cytokine stimulation. However, consistent with our previous results, STAT1 and STAT3 have been found to be present in nuclei independently of tyrosine phosphorylation, in a cell type-specific manner, and the nuclear import mechanism of U-STAT1 is completely distinct from that of phosphorylated STAT1 (13, 14). The nuclear import of tyrosinephosphorylated STAT1 dimers dep...

The activation of STAT3 by tyrosine phosphorylation, essential for normal development and for a normal inflammatory response to invading pathogens, is kept in check by negative regulators. Abnormal constitutive activation of STAT3, which contributes to the pathology of cancer and to chronic inflammatory diseases such as rheumatoid arthritis, occurs when negative regulation is not fully effective. SOCS3, the major negative regulator of STAT3, is induced by tyrosine-phosphorylated STAT3 and terminates STAT3 phosphorylation about 2 h after initial exposure of cells to members of the IL-6 family of cytokines by binding cooperatively to the common receptor subunit gp130 and JAKs 1 and 2. We show here that when the epidermal growth factor receptor (EGFR) is present and active, STAT3 is rephosphorylated about 4 h after exposure of cells to IL-6 or oncostatin M and remains active for many hours. Newly synthesized IL-6 drives association of the IL-6 receptor and gp130 with EGFR, leading to EGFR-dependent rephosphorylation of STAT3, which is not inhibited by the continued presence of SOCS3. This second wave of STAT3 activation supports sustained expression of a subset of IL-6-induced proteins, several of which play important roles in inflammation and cancer, in which both IL-6 secretion and EGFR levels are often elevated.A fter ligand-induced dimerization of the IL-6 receptor (IL-6R), the associated kinases JAK1 and JAK2 cross-phosphorylate tyrosine residues of adjacent glycoprotein 130 (gp130) subunits of the complex. The SH2 domain of STAT3 binds to newly phosphorylated tyrosines, followed by the phosphorylation of Y705 of STAT3 (1). Two phosphorylated STAT3 monomers then dimerize and translocate to the nucleus,

Constitutive expression of interferons (IFNs) and activation of their signaling pathways have pivotal roles in host responses to malignant cells in the tumor microenvironment. IFNs are induced from the innate immune system and in tumors through stimulation of Toll-like receptors (TLRs) and through other signaling pathways in response to specific cytokines. Although in the oncologic context IFNs have been thought of more as exogenous pharmaceuticals, the autocrine and paracrine actions of endogenous IFNs probably have even more critical effects on neoplastic disease outcomes. Through high-affinity cell surface receptors, IFNs modulate transcriptional signaling, leading to regulation of over 2000 genes with varying patterns of temporal expression. Induction of the gene products by both unphosphorylated and phosphorylated STAT1 after ligand binding, results in alterations in tumor cell survival, inhibition of angiogenesis, and augmentation of actions of T, natural killer (NK), and dendritic cells. The interferon-stimulated gene (ISG) signature can be a favorable biomarker of immune response but, in a seemingly paradoxical finding, a specific subset of the full ISG signature indicates an unfavorable response to DNA damaging interventions such as radiation. IFNs in the tumor microenvironment thus can alter the emergence, progression, and regression of malignancies.

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