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Regulation of interferon-inducible proteins by doxorubicin via IFN-alpha-JAK-STAT signaling in tumor cells

ABSTRACT: Activation of the immune system is a way for host tissue to defend itself against tumor growth. Hence, treatment strategies that are based on immunomodulation are on the rise. Conventional cytostatic drugs such as the anthracycline doxorubicin can also activate immune cell functions of macrophages and natural killer cells. In addition, cytotoxicity of doxorubicin can be enhanced by combining this drug with the cytokine IFN-alpha. Although doxorubicin is one of the most applied cytostatics, the molecular mechanisms of its immunomodulation ability are not investigated thoroughly. In microarray analyses of HeLa cells, a set of 19 genes related to interferon signaling was significantly overrepresented among genes regulated by doxorubicin exposure including STAT-1, -2, IRF9, NMI, and caspase 1. Regulation of these genes by doxorubicin was verified with Real-Time PCR and immunoblotting. An enhanced secretion of IFN-alpha was observed when HeLa cells were exposed to doxorubicin as compared to untreated cells. IFN-alpha neutralizing antibodies and inhibitors of JAK-STAT signaling (ATA and AG490) significantly abolished doxorubicin-stimulated expression of interferon signaling-related genes. Furthermore, inhibition of JAK-STAT signaling significantly reduced doxorubicin induced caspase 3 activation and desensitized HeLa cells to doxorubicin cytotoxicity. In conclusion, we demonstrate that doxorubicin induces interferon-responsive genes via IFN-alpha-JAK-STAT1 signaling and that this pathway is relevant for doxorubicin’s cytotoxicity in HeLa cells. As immunomodulation is a promising strategy in anticancer treatment, this novel mode of action of doxorubicin may help to further improve the use of this drug among different types of anticancer treatment strategies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE30988 | GEO | 2012/05/23

SECONDARY ACCESSION(S): PRJNA146203

REPOSITORIES: GEO

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Project description:Maier2022 - Stochastic Dynamics of Type I Interferon Responses Our study aims to determine whether and how biochemical noise affects the information transduced in the JAK-STAT signaling pathway and investigate the transition between basal and activated state. To this end, we studied the stochastic responses of MxA and IFIT1 expression in Huh7.5 cells stimulated with IFN-$\alpha$. Using fluorescent reporters under the control of the authentic promoter/enhancer region of IFIT1 and MxA we collected data displaying the differences between expressing and non-expressing cells for the marker genes in a time-course experiment. We hypothesize that the JAK-STAT signaling pathway efficiently transmits information under stochastic environments. To test our working hypothesis, we developed a detailed mathematical model using the obtained time-resolved flow cytometry data to describe the elements in the JAK-STAT signaling pathway at single-cell resolution. This model allowed us to systematically test the influence of intrinsic and extrinsic noise in the IFN response. The developed model consists of 42 species and 62 reactions (reactions m1 to m62). To name the variables in the model we used the following conventions: 1) variables referring to mRNA are denoted by $m$ prefix. 2) Variables in phosphorylated use $p$ as prefix. 3) Gene promoters are represented by the gene's name in lowercase. 4) R1, R2, IR, AR and RC, represent the IFN receptor subunits, inactive, active and complex forms, respectively. 5) The compartment is superscripted to the species if the species exist in multiple compartments. A graphical representation of the interaction between variables in the model is given in Maier et. al 2022 (Fig. 1) and all reactions are listed in the Supplementary Information, Section S2.1. Note that we publish the SBML model without the observables (e.g. exp_IRF9_n) as the change in the number of particles is defined in relation to the starting value in the COPASI model which is not supported in SBML format. In order to reproduce the parameter estimation without any extra worj, we recommend the direct download of the provided copasi model linked in the article. This model is described in the article: Stochastic Dynamics of Type I Interferon Responses Benjamin D. Maier(*), Luis U. Aguilera(*), Sven Sahle, Pascal Mutz, Priyata Kalra, Christopher Dächert, Ralf Bartenschlager, Marco Binder, Ursula Kummer PLOS Computational Biology, 2022 (*) Equally contributing authors Abstract: Interferon (IFN) activates the transcription of several hundred of IFN stimulated genes (ISGs) that constitute a highly effective antiviral defense program. Cell-to-cell variability in the induction of ISGs is well documented, but its source and effects are not completely understood. The molecular mechanisms behind this heterogeneity have been related to randomness in molecular events taking place during the JAK-STAT signaling pathway. Here, we study the sources of variability in the induction of the IFN-alpha response by using MxA and IFIT1 activation as read-out. To this end, we integrate time-resolved flow cytometry data and stochastic modeling of the JAK-STAT signaling pathway. The complexity of the IFN response was matched by fitting probability distributions to time-course flow cytometry snapshots. Both, experimental data and simulations confirmed that the MxA and IFIT1 induction circuits generate graded responses rather than all-or-none responses. Subsequently, we quantify the size of the intrinsic variability at different steps in the pathway. We found that stochastic effects are transiently strong during the ligand-receptor activation steps and the formation of the ISGF3 complex, but negligible for the final induction of the studied ISGs. We conclude that the JAK-STAT signaling pathway is a robust biological circuit that efficiently transmits information under stochastic environments.

Project description:Interferon-alpha (pegylated interferon and ribavirin) is used as standard-of-care therapeutic for chronic hepatitis C virus infection. Besides good cure in some patients other patients do not benefit from the treatment dependent on the virus type and host factors. One class of putative effector proteins is the family of Suppressors of cytokine signalling (SOCS). They act in a classical negative feedback-loop against the action of interferons and many other cytokines. It has been proven that some of them, in particular SOCS1 and SOCS3, inhibit the expression of interferon induced antiviral proteins. Their mode of action depends on the signal they are interfering with. In relation to the interferon-gamma pathway, they are thought to act on the interferon-alpha receptors by masking its recognition site for the Janus kinases (JAK), by blocking the kinase activity of the JAKs and coincidentally hindering STAT molecules from binding to the kinases. They are also thought to ubiquitinate the JAKs resulting in their proteosomal degradation. The function of SOCS proteins in suppressing the interferon-alpha pathway has not yet been characterized exhaustively. This study should unveil links to understand the resistance in interferon-alpha therapy. As results we got almost complete silencing of JAK-STAT signaling in SOCS1 over-expressing cells and tissue-dependent partially suppressed gene induction in SOCS3 over-expressing cell lines. Two human cancer cell lines (ME-15, HuH-7) were stably transfected with pcDNA3.1-SOCS plasmids in presence of geneticin and daughter cell lines were generated after singularization of cells. Next, original cell lines as well as SOCS1 and SOCS3 over-expressing cell lines were treated with 1000 U/ml interferon-alpha for 4 or 24 hours or in normal culture medium. Cells lines obtained from SOCS4 plasmid transfections were screened as additional control. Gene expression levels of cell cultured in control (0 for 4 hours, 2 for 24 hours) or interferon-alpha supplemented medium for 4 hours (1) or 24 hours (4) were analyzed. mRNA abundance was measured in triplicates using 12x8-sample commercial Illumina microarrays (HumanRef 8, version 3) and scanner system (iScan) as well as reagents recommend by Illumina (IlluminaM-BM-. TotalPrep Kit).

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Regulation of interferon-inducible proteins by doxorubicin via IFN-alpha-JAK-STAT signaling in tumor cells

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