Type I interferon (v affect cell differentiation, proliferation, and survival in nearly all kinds of cell types, and are efficacious in the treatment of malignancies such as chronic myeloid leukemia. In recent years, the usage of IFN? for solid cancer chemotherapy has received considerable attention. IFN?-activated STAT2, STAT1, and IRF9 form the transcription factor IFN-stimulated gene factor-3 (ISGF3) complex, which binds to interferon stimulated response element (ISRE) sequence for transcriptional activation. IFN? activated STAT proteins also form homo- or hetero-dimer that bind to sis-inducible element (SIE) to regulate gene expression. IFN? receptor (IFN?R) consist two cognate subunits: IFN?R1 and IFN?R2. IFN? binds IFN?R2 to instigate its association with IFN?R1. IFN?R1 and IFN?R2 are cross-phosphorylated by their associated tyrosine kinases (Jak1 and Tyk2), followed by subsequent STAT2 and STAT1 recruitment and phosphorylational activation. The mechanism of how IFN? activates IRF9 is unknown. IRF9-dependent transcriptional activation can be enhanced by treating the cells with deacetylase inhibitors, suggesting IRF9 activation involves acetylation. We detected IFN?R2 acetylation by recruiting tumor suppressor-like transcription cofactor CREB-binding protein (CBP) or its homologous p300. Acetylated IFN?R2 can then recruit IRF9. IRF9 as well as STAT2 and STAT1 are all acetylated by CBP prior to forming the transcriptional active ISGF3 complex. IFN?R also recruits deacetylases including SIRT and HDAC members. Although tyrosine phosphorylation has long been accepted to play a paramount role in cytokine receptor signal transduction, our findings based upon preliminary data and protein secondary structural analysis challenge this concept. The overarching hypothesis to be tested here is that CBP/p300-mediated acetylation cascade triggered by IFN? plays an indispensable role in signal transduction for anti-proliferation, proapotosis, and anti-metastasis gene regulation. Disrupting IFN?R and deacetylase association with deacetylase inhibitors may improve the therapeutic effects of IFN? in cancer. We will apply site-directed mutagenesis, FRET technology, as well as the antibody array technology developed in our lab in order to (1) define the acetyltransferase activity associated with CBP/p300 on IFN?R activation in intracellular signaling;(2) analyze IFN?R, STAT1 and ISGF3 deacetylation by HDAC or SIRT in signal termination;and (3) determine whether acetylated STAT1 dimer or ISGF3 complex is critical for IFN?- mediated anti-proliferation, proapoptosis, and antimetastasis gene regulation in cancer cells. These approaches will help elucidate the function of acetylation and deacetylation in the regulation of IFN?R activation, STAT dimer, and ISGF3 complex formation for signal transduction and transcription, and allow us to maximize the therapeutic applications of IFN? for cancer.
ABSTRACT NARRATIVE Type I interferons (IFN,) have been widely used for treatment of cancer and virus-infected diseases. Our recent findings suggest that IFN-alpha can have a previous totally unknown signaling event, i.e., lysine acetylation cascade, leading to anti-proliferation and anti-viral gene activation. In this proposal, we want to address in detail, the precise mechanism of how acetylation cascade works in turning on genes involved in anti-proliferation and anti-metastasis in response to IFN treatment in cancer cells.
