Anthony J. Scarzello

Interferon: cellular executioner or white knight?

Interferons (IFNs) are a family of pleiotropic cytokines that typically exhibit antiviral, antiproliferative, antitumor, and immunomodulatory properties. While their complex mechanisms of action remain unclear, IFNs are used clinically in the treatment of viral infections, such as hepatitis B and hepatitis C, and remain the primary treatment for a limited number of malignancies, such as melanoma, hairy cell leukemia, and non-Hodgkins lymphoma and in autoimmune diseases such as multiple sclerosis. IFNs not only regulate somatic cell growth and division but also influence cell survival through the modulation of apoptosis. Paradoxically, IFNs are described to be both pro- and anti-apoptotic in nature. The biological effects of IFNs are primarily mediated via activation of the JAK/STAT pathway, formation of the ISGF3 and STAT1:STAT1 protein complexes, and the subsequent induction of IFN-stimulated genes. However, the activation of JAK/STAT-independent signal transduction pathways also contribute to IFN-mediated responses. To further demonstrate the complexity of the downstream events following stimulation, oligonucleotide microarray studies have shown that in excess of 300 genes are induced following treatment with IFN, some of which are crucial to the induction of apoptosis and cell growth control. In this review we describe the recent advances made in elucidating the various signaling pathways that are activated by IFNs and how these diverse signals contribute to the regulation of cell growth and apoptosis and inhibition of viral replication. Furthermore, we highlight the role of specific signaling molecules and the function(s) of particular IFN-stimulated genes that have been implicated in determining cell fate in response to IFN, as well as the clinical experience of IFN immunotherapy.

IFN-α and IFN-λ differ in their antiproliferative effects and duration of JAK/STAT signaling activity

Interferon (IFN)-λ, also known as IL-28A, IL-28B or IL-29, is a new type III IFN, which like type I IFN-(α/β), activates common elements of the JAK/STAT signaling pathway and exhibits antiproliferative activity. Currently, IFN-α is used in the treatment of certain forms of cancer, but its antitumor effects are limited and associated with high toxicity. In this study, we determined whether IFN-λ induced the same level of cell growth inhibition relative to IFN-α. To this effect HaCaT cells, which are typically growth inhibited by IFN-α, underwent apoptosis in response to IFN-λ. Next, in contrast to IFN-α stimulation, IFN-λ prolonged the duration of activated STAT1 and STAT2. Furthermore, the kinetics of IFN-stimulated genes was different as IFN-λ induced a delayed but stronger induction of IFN-responsive genes. Components of the JAK/STAT pathway remained essential for the antiproliferative effects of IFN-α and IFN-λ. IFN-λ-induced persistence of STAT activation required de novo protein synthesis and was in part due to a delay in STAT2 inactivation. Thus our data demonstrate that the duration of IFN-λ signaling is different from that of IFN-α, and that IFN-λ could be a suitable cytokine to evaluate for cancer therapy.

Chronic inflammation, immune escape, and oncogenesis in the liver: a unique neighborhood for novel intersections.

Sustained hepatic inflammation, driven by alcohol consumption, nonalcoholic fatty liver disease, and/or chronic viral hepatitis (hepatitis B and C), results in damage to parenchyma, oxidative stress, and compensatory regeneration/proliferation. There is substantial evidence linking these inflammation‐associated events with the increased incidence of hepatocellular carcinogenesis. Although acute liver inflammation can play a vital and beneficial role in response to liver damage or acute infection, the effects of chronic liver inflammation, including liver fibrosis and cirrhosis, are sufficient in a fraction of individuals to initiate the process of transformation and the development of hepatocellular carcinoma. This review highlights immune‐dependent mechanisms that may be associated with hepatocellular oncogenesis, including critical transformative events/pathways in the context of chronic inflammation and subverted tolerogenesis. (HEPATOLOGY 2012)

Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediated mitigation of an immunosuppressive tumor microenvironment

Treatment of mice bearing orthotopic, metastatic tumors with anti-CD40 antibody resulted in only partial, transient anti-tumor effects whereas combined treatment with IL-2/anti-CD40, induced tumor regression. The mechanisms for these divergent anti-tumor responses were examined by profiling tumor-infiltrating leukocyte subsets and chemokine expression within the tumor microenvironment after immunotherapy. IL-2/anti-CD40, but not anti-CD40 alone, induced significant infiltration of established tumors by NK and CD8+ T cells. To further define the role of chemokines in leukocyte recruitment into tumors, we evaluated anti-tumor responses in mice lacking the chemokine receptor, CCR2. The anti-tumor effects and leukocyte recruitment mediated by anti-CD40 alone, were completely abolished in CCR2−/− mice. In contrast, IL-2/anti-CD40-mediated leukocyte recruitment and reductions in primary tumors and metastases were maintained in CCR2−/− mice. Treatment of mice with IL-2/anti-CD40, but not anti-CD40 alone, also caused an IFN-γ-dependent increase in the expression of multiple Th1 chemokines within the tumor microenvironment. Interestingly, although IL-2/anti-CD40 treatment increased Tregs in the spleen, it also caused a coincident IFN-γ-dependent reduction in CD4+/FoxP3+ Tregs, myeloid-derived suppressor cells and Th2 chemokine expression specifically within the tumor microenvironment that was not observed after treatment with anti-CD40 alone. Similar effects were observed using IL-15 in combination with anti-CD40. Taken together, our data demonstrate that IL-2/anti-CD40, but not anti-CD40 alone, can preferentially reduce the overall immunosuppressive milieu within the tumor microenvironment. These results suggest that the use of anti-CD40 in combination with IL-2 or IL-15 may hold substantially more promise for clinical cancer treatment than anti-CD40 alone.

Coactivation of AKT and β-catenin in mice rapidly induces formation of lipogenic liver tumors.

Obesity is a risk factor for development of certain cancers but the basis for this risk is unclear. In this study, we developed a novel mouse model that demonstrates directly how lipogenic phenotypes commonly associated with diet-induced metabolic syndromes can influence hepatic cancer development. Activated AKT and β-catenin (AKT/CAT) genes were hydrodynamically codelivered using the Sleeping Beauty transposon to initiate liver tumorigenesis. AKT/CAT and MET/CAT combination induced microscopic tumor foci by 4 weeks, whereas no tumorigenesis resulted from delivery of AKT, MET, or CAT alone. Primary AKT/CAT tumor cells were steatotic (fatty) hepatocellular adenomas which progressed to hepatocellular carcinomas (HCC) upon in vivo passage, whereas primary MET/CAT tumors emerged directly as frank HCC. Conversion of AKT/CAT tumor cells to frank HCC during passage was associated with induction of the human HCC marker α-fetoprotein and the stem cell marker CD133. Using hierarchical clustering and gene set enrichment analysis, we compared the primary murine AKT/CAT and MET/CAT tumors to a panel of 53 human HCCs and determined that these two mouse models could be stratified as distinct subtypes associated in humans with poor clinical prognosis. The chief molecular networks identified in primary and passaged AKT/CAT tumors were steatosis and lipid metabolic pathways, respectively. Our findings show how coactivation of the AKT and CAT pathways in hepatocytes can efficiently model development of a lipogenic tumor phenotype. Furthermore, we believe that our approach could speed the dissection of microenvironmental factors responsible for driving steatotic-neoplastic transformation to frank carcinoma, through genetic modification of existing immunodefined transgenic models.

STAT2 contributes to promotion of colorectal and skin carcinogenesis.

Signal transducer and activator of transcription 2 (STAT2) is an essential transcription factor in the type I IFN (IFN-α/β) signal transduction pathway and known for its role in mediating antiviral immunity and cell growth inhibition. Unlike other members of the STAT family, IFNs are the only cytokines known to date that can activate STAT2. Given the inflammatory and antiproliferative dual nature of IFNs, we hypothesized that STAT2 prevents inflammation-induced colorectal and skin carcinogenesis by altering the inflammatory immune response. Contrary to our hypothesis, deletion of STAT2 inhibited azoxymethane/dextran sodium sulfate–induced colorectal carcinogenesis as measured by prolonged survival, lower adenoma incidence, smaller polyps, and less chronic inflammation. STAT2 deficiency also inhibited 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate–induced skin carcinogenesis as indicated by reduced papilloma multiplicity. A potential mechanism by which STAT2 promotes carcinogenesis is through activation of proinflammatory mediators. Deletion of STAT2 decreased azoxymethane/dextran sodium sulfate–induced expression and release of proinflammatory mediators, such as interleukin-6 and CCL2, and decreased interleukin-6 release from skin carcinoma cells, which then decreased STAT3 activation. Our findings identify STAT2 as a novel contributor to colorectal and skin carcinogenesis that may act to increase the gene expression and secretion of proinflammatory mediators, which in turn activate the oncogenic STAT3 signaling pathway. Cancer Prev Res; 3(4); 495–504. ©2010 AACR.

Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2

Type I IFNs (IFN-α/β) are pleitropic cytokines widely used in the treatment of certain malignancies, hepatitis B and C, and multiple sclerosis. IFN resistance is a challenging clinical problem to overcome. Hence, understanding the molecular mechanism by which IFN immunotherapy ceases to be effective is of translational importance. In this study, we report that continuous IFN-α stimulation of the human Jurkat variant H123 led to resistance to type I IFN–induced apoptosis due to a loss of signal transducers and activators of transcription 2 (STAT2) expression. The apoptotic effects of IFN-α were hampered as STAT2-deficient cells were defective in activating the mitochondrial-dependent death pathway and ISGF3-mediated gene activation. Reconstitution of STAT2 restored the apoptotic effects of IFN-α as measured by the loss of mitochondrial membrane potential, cytochrome c release from mitochondria, caspase activation, and ultimately cell death. Nuclear localization of STAT2 was a critical event as retention of tyrosine-phosphorylated STAT2 in the cytosol was not sufficient to activate apoptosis. Furthermore, silencing STAT2 gene expression in Saos2 and A375S.2 tumor cell lines significantly reduced the apoptotic capacity of IFN-α. Altogether, we show that STAT2 is a critical mediator in the activation of type I IFN–induced apoptosis. More importantly, defects in the expression or nuclear localization of STAT2 could lessen the efficacy of type I IFN immunotherapy. Mol Cancer Res; 8(1); 80–92

Stat2 loss leads to cytokine-independent, cell-mediated lethality in LPS-induced sepsis

Deregulated Toll-like receptor (TLR)-triggered inflammatory responses that depend on NF-κB are detrimental to the host via excessive production of proinflammatory cytokines, including TNF-α. Stat2 is a critical component of type I IFN signaling, but it is not thought to participate in TLR signaling. Our study shows that LPS-induced lethality in Stat2−/− mice is accelerated as a result of increased cellular transmigration. Blocking intercellular adhesion molecule-1 prevents cellular egress and confers survival of Stat2−/− mice. The main determinant of cellular egress in Stat2−/− mice is the genotype of the host and not the circulating leukocyte. Surprisingly, lethality and cellular egress observed on Stat2−/− mice are not associated with excessive increases in classical sepsis cytokines or chemokines. Indeed, in the absence of Stat2, cytokine production in response to multiple TLR agonists is reduced. We find that Stat2 loss leads to reduced expression of NF-κB target genes by affecting nuclear translocation of NF-κB. Thus, our data reveal the existence of a different mechanism of LPS-induced lethality that is independent of NF-κB triggered cytokine storm but dependent on cellular egress.

TCR-dependent and –independent activation underlie liver-specific regulation of NKT cells

The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.

TNFR2 expression by CD4 effector T cells is required to induce full-fledged experimental colitis

There is now compelling evidence that TNFR2 is constitutively expressed on CD4+ Foxp3+ regulatory T cells (Tregs) and TNF-TNFR2 interaction is critical for the activation, expansion and functional stability of Tregs. However, we showed that the expression of TNFR2 was also up-regulated on CD4+ Foxp3− effector T cells (Teffs) upon TCR stimulation. In order to define the role of TNFR2 in the pathogenic CD4 T cells, we compared the effect of transferred naïve CD4 cells from WT mice and TNFR2−/− mice into Rag 1−/− recipients. Transfer of TNFR2-deficient Teff cells failed to induce full-fledged colitis, unlike WT Teffs. This was due to defective proliferative expansion of TNFR2-deficient Teff cells in the lymphopenic mice, as well as their reduced capacity to express proinflammatory Th1 cytokine on a per cell basis. In vitro, the proliferative response of TNFR2 deficient naïve CD4 cells to anti-CD3 stimulation was markedly decreased as compared with that of WT naïve CD4 cells. The hypoproliferative response of TNFR2-deficient Teff cells to TCR stimulation was associated with an increased ratio of p100/p52, providing a mechanistic basis for our findings. Therefore, this study clearly indicates that TNFR2 is important for the proliferative expansion of pathogenic Teff cells.