Erin E. Childs

Erythropoietin-mediated activation of JAK-STAT signaling contributes to cellular invasion in head and neck squamous cell carcinoma

Originally characterized as a growth factor for erythrocytes, erythropoietin (EPO) is used to treat anemia and fatigue in cancer patients receiving radiation therapy and chemotherapy. EPO and the EPO receptor (EPOR) are expressed in nonhematopoietic cells and cancers. However, the role of EPO and EPOR within nonhematopoietic cancer cells remains incompletely understood. Although a recent clinical trial demonstrated worse tumor control and survival in head and neck cancer patients treated with EPO, the role of EPO and EPOR in head and neck squamous cell carcinoma (HNSCC) has not been examined. In the present study, we demonstrate the previously unrecognized EPO-mediated invasion by HNSCC cells through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. Furthermore, we confirmed the expression of EPO and EPOR in a panel of human HNSCC cell lines and tissue specimens. Pharmacological doses of EPO also had a limited proliferation effect in these cell lines. These results define a novel role for EPO in mediating tumor cell invasion. Increased levels of EPO and EPOR in lymph node metastases as compared to primary tumors from HNSCC patients further support the role of EPO/EPOR in HNSCC disease progression and metastasis.

IL-17RC Is Required for Immune Signaling via an Extended SEF/IL-17R Signaling Domain in the Cytoplasmic Tail

IL-17 mediates essential inflammatory responses in host defense and autoimmunity. The IL-17A–IL-17F signaling complex is composed of IL-17RA and IL-17RC, both of which are necessary for signal transduction. To date, the specific contribution of IL-17RC to downstream signaling remains poorly understood. To define the regions within the IL-17RC cytoplasmic tail required for signal transduction, we assayed signaling by a panel of IL-17RC deletion mutants. These findings reveal that IL-17RC inducibly associates with a specific glycosylated IL-17RA isoform, in a manner independent of the IL-17RC cytoplasmic tail. Using expression of the IL-17 target genes IL-6 and 24p3/lipocalin-2 as a readout, functional reconstitution of signaling in IL-17RC−/− fibroblasts required the SEF/IL-17R signaling domain (SEFIR), a conserved motif common to IL-17R family members. Unexpectedly, the IL-17RC SEFIR alone was not sufficient to reconstitute IL-17–dependent signaling. Rather, an additional sequence downstream of the SEFIR was also necessary. We further found that IL-17RC interacts directly with the adaptor/E3 ubiquitin ligase Act1, and that the functional IL-17RC isoforms containing the extended SEFIR region interact specifically with a phosphorylated isoform of Act1. Finally, we show that IL-17RC is required for in vivo IL-17–dependent responses during oral mucosal infections caused by the human commensal fungus Candida albicans. These results indicate that IL-17RC is vital for IL-17–dependent signaling both in vitro and in vivo. Insight into the mechanisms by which IL-17RC signals helps shed light on IL-17–dependent inflammatory responses and may ultimately provide an avenue for therapeutic intervention in IL-17–mediated diseases.

Antiproliferative Mechanisms of a Transcription Factor Decoy Targeting Signal Transducer and Activator of Transcription (STAT) 3: The Role of STAT1

We previously developed a transcription factor decoy targeting signal transducer and activator of transcription 3 (STAT3) and reported antitumor activity in both in vitro and in vivo models of squamous cell carcinoma of the head and neck (SCCHN). Based on the known existence of STAT1-STAT3 heterodimers, the high sequence homology between STAT1 and STAT3, as well as expression of both STAT1 and STAT3 in SCCHN, we examined whether the STAT3 decoy interferes with STAT1 signaling. SCCHN cell lines with different STAT1 expression levels (but similar STAT3 levels) were used. Both cell lines were sensitive to the growth-inhibitory effects of the STAT3 decoy compared with a mutant control decoy. Intact STAT1 signaling was demonstrated by interferon-γ (IFN-γ)-mediated induction of STAT1 phosphorylation (Tyr701) and interferon-regulatory factor-1 (IRF-1) expression. Treatment with the STAT3 decoy (but not a mutant control decoy) resulted in inhibition of IRF-1 protein expression in both cell lines, indicating specific inhibition of STAT1 signaling by the STAT3 decoy. Because STAT1 is a potential tumor suppressor, we also investigated whether STAT1 signaling mitigated the therapeutic efficacy of the STAT3 decoy. In both PCI-15B and UM-22B cells, STAT1 siRNA treatment resulted in decreased STAT1 expression, without altering the antitumor activity of the STAT3 decoy. Likewise, the antitumor effects of the STAT3 decoy were not altered by STAT1 activation upon IFN-γ treatment. These results suggest that the therapeutic mechanisms of STAT3 blockade using a transcription factor decoy are independent of STAT1 activation.

Intratumoral Epidermal Growth Factor Receptor Antisense DNA Therapy in Head and Neck Cancer: First Human Application and Potential Antitumor Mechanisms

PURPOSE Squamous cell carcinoma of the head and neck (SCCHN) is characterized by upregulation of the epidermal growth factor receptor (EGFR). We developed a novel strategy to target EGFR by using a therapeutic gene that consisted of an EGFR antisense (AS) gene sequence under U6 promoter control. A phase I clinical trial was conducted to evaluate the safety and biologic effects of EGFR AS. PATIENTS AND METHODS Patients with advanced SCCHN who were refractory to standard therapies and who had at least one assessable and accessible lesion were enrolled. The EGFR AS dose was escalated in successive cohorts (six dose levels; 60 to 1,920 microg/injection). Patients received four weekly intratumoral EGFR AS injections. Tumor biopsies were performed before and after completion of therapy. Treatment response was assessed by tumor volume measurements (positron emission tomography/computed tomography), and levels of target proteins were assessed by immunohistochemistry. RESULTS Seventeen assessable patients were treated. No grades 3 to 4 or dose-limiting toxicities were noted, and a maximum-tolerated dose was not reached. Five patients (29%) achieved a clinical response, which included two complete responses (CRs) and three partial responses (PRs); two additional patients had stable disease (SD) as the best response. Patients with disease control (CR + PR + SD) had tumors with higher EGFR and lower STAT3 expression at baseline compared with patients who had progressive disease (P = .0312 and P = .095, respectively). CONCLUSION Intratumoral EGFR AS was safe and resulted in antitumor activity in patients with advanced SCCHN. Baseline levels of high EGFR and low STAT3 may be associated with antitumor effects.

C/EBPβ Promotes Immunity to Oral Candidiasis through Regulation of β-Defensins.

Humans or mice subjected to immunosuppression, such as corticosteroids or anti-cytokine biologic therapies, are susceptible to mucosal infections by the commensal fungus Candida albicans. Recently it has become evident that the Th17/IL-17 axis is essential for immunity to candidiasis, but the downstream events that control immunity to this fungus are poorly understood. The CCAAT/Enhancer Binding Protein-β (C/EBPβ) transcription factor is important for signaling by multiple inflammatory stimuli, including IL-17. C/EBPβ is regulated in a variety of ways by IL-17, and controls several downstream IL-17 target genes. However, the role of C/EBPβ in vivo is poorly understood, in part because C/EBPβ-deficient mice are challenging to breed and work with. In this study, we sought to understand the role of C/EBPβ in the context of an IL-17-dependent immune response, using C. albicans infection as a model system. Confirming prior findings, we found that C/EBPβ is required for immunity to systemic candidiasis. In contrast, C/EBPβ-/- mice were resistant to oropharyngeal candidiasis (OPC), in a manner indistinguishable from immunocompetent WT mice. However, C/EBPβ-/- mice experienced more severe OPC than WT mice in the context of cortisone-induced immunosuppression. Expression of the antimicrobial peptide β-defensin (BD)-3 correlated strongly with susceptibility in C/EBPβ-/- mice, but no other IL-17-dependent genes were associated with susceptibility. Therefore, C/EBPβ contributes to immunity to mucosal candidiasis during cortisone immunosuppression in a manner linked to β-defensin 3 expression, but is apparently dispensable for the IL-17-dependent response.

IL-17 integrates multiple self-reinforcing, feed-forward mechanisms through the RNA binding protein Arid5a

The RNA binding protein Arid5a augments both mRNA stability and translation to promote cellular responses to IL-17. Stabilizing the IL-17 response The inflammatory cytokine IL-17 can stimulate both antifungal host defense and autoimmunity by promoting the stability of target mRNAs (see the Focus by Puel and Casanova). Amatya et al. found that IL-17 increased the abundance of the RNA binding protein Arid5a in mouse cells. Loss of Arid5a decreased the cellular response to IL-17 by reducing the mRNA stability of a selection of IL-17–stimulated transcripts. For others, Arid5a instead interacted with the translation initiation factor eIF4G to augment their translation. Thus, Arid5a uses multiple posttranscriptional mechanisms to enhance IL-17 signaling. Interleukin-17A (IL-17A) not only stimulates immunity to fungal pathogens but also contributes to autoimmune pathology. IL-17 is only a modest activator of transcription in experimental tissue culture settings. However, IL-17 controls posttranscriptional events that enhance the expression of target mRNAs. Here, we showed that the RNA binding protein (RBP) Arid5a (AT-rich interactive domain-containing protein 5a) integrated multiple IL-17–driven signaling pathways through posttranscriptional control of mRNA. IL-17 induced expression of Arid5a, which was recruited to the adaptor TRAF2. Arid5a stabilized IL-17–induced cytokine transcripts by binding to their 3′ untranslated regions and also counteracted mRNA degradation mediated by the endoribonuclease MCPIP1 (Regnase-1). Arid5a inducibly associated with the eukaryotic translation initiation complex and facilitated the translation of the transcription factors (TFs) IκBζ (Nfkbiz ) and C/EBPβ (Cebpb). These TFs in turn transactivated IL-17–dependent promoters. Together, these data indicated that Arid5a orchestrates a feed-forward amplification loop, which promoted IL-17 signaling by controlling mRNA stability and translation.

IL-17 Signaling triggers degradation of the constitutive NF-κB inhibitor ABIN-1

IL-17 activates NF-κB and induces expression of proinflammatory genes. IL-17 drives disease in autoimmune conditions, and anti–IL-17 Abs have shown impressive success in the clinic. Although produced by lymphocytes, IL-17 predominantly signals in fibroblasts and epithelial cells. IL-17–driven inflammation is kept in check by negative feedback signaling molecules, including the ubiquitin editing enzyme A20, whose gene TNFAIP3 is linked to autoimmune disease susceptibility. The A20 binding inhibitor of NF-κB activation 1 (ABIN-1) is an A20-binding protein encoded by the TNIP1 gene, which is also linked to autoimmune disease susceptibility including psoriasis. Accordingly, we hypothesized that ABIN-1 might play a role in negatively regulating IL-17 signaling activity. Indeed, ABIN-1 enhanced both tonic and IL-17–dependent NF-κB signaling in IL-17–responsive fibroblast cells. Interestingly, the inhibitory activities of ABIN-1 on IL-17 signaling were independent of A20. ABIN-1 is a known NF-κB target gene, and we found that IL-17–induced activation of NF-κB led to enhanced ABIN-1 mRNA expression and promoter activity. Surprisingly, however, the ABIN-1 protein was inducibly degraded following IL-17 signaling in a proteasome-dependent manner. Thus, ABIN-1, acting independently of A20, restricts both baseline and IL-17–induced inflammatory gene expression. We conclude that IL-17–induced signals lead to degradation of ABIN-1, thereby releasing a constitutive cellular brake on NF-κB activation.

CCAAT/Enhancer-binding protein β promotes pathogenesis of EAE

HIGHLIGHTSCebpb−/− mice are resistant to EAE.In vivo Th17 responses are impaired in absence of C/EBP&bgr;.Th17 differentiation is normal in APC‐free cultures.C/EBP&bgr; binds to the IL‐23R in myeloid and Th17 cells. ABSTRACT The CCAAT/Enhancer Binding Protein &bgr; (C/EBP&bgr;) transcription factor is activated by multiple inflammatory stimuli, including IL‐17 and LPS, and C/EBP&bgr; itself regulates numerous genes involved in inflammation. However, the role of C/EBP&bgr; in driving autoimmunity is not well understood. Here, we demonstrate that Cebpb−/− mice are resistant to EAE. Cebpb−/− mice exhibited reduced lymphocyte and APC infiltration into CNS following EAE induction. Furthermore, MOG‐induced Th17 cytokine production was impaired in draining LN, indicating defects in Th17 cell priming. In vitro Th17 polarization studies indicated that T cell responses are not inherently defective, instead supporting the known roles for C/EBP&bgr; in myeloid lineage cell activation as the likely mechanism for defective Th17 priming in vivo. However, we did uncover an unexpected role for C/EBP&bgr; in regulating ll23r expression in APCs. ChIP assays confirmed that C/EBP&bgr; binds directly to the Il23r gene promoter in dendritic cells and Th17 cells. These data establish C/EBP&bgr; as a key driver of autoimmune inflammation in EAE, and propose a novel role for C/EBP&bgr; in regulation of IL‐23R expression.

A Candida albicans Strain Expressing Mammalian Interleukin-17A Results in Early Control of Fungal Growth during Disseminated Infection

ABSTRACT Candida albicans is normally a commensal fungus of the human mucosae and skin, but it causes life-threatening systemic infections in hospital settings in the face of predisposing conditions, such as indwelling catheters, abdominal surgery, or antibiotic use. Immunity to C. albicans involves various immune parameters, but the cytokine interleukin-17A (IL-17A) (also known as IL-17) has emerged as a centrally important mediator of immune defense against both mucosal and systemic candidiasis. Conversely, IL-17A has been suggested to enhance the virulence of C. albicans, indicating that it may exert detrimental effects on pathogenesis. In this study, we hypothesized that a C. albicans strain expressing IL-17A would exhibit reduced virulence in vivo. To that end, we created a Candida-optimized expression cassette encoding murine IL-17A, which was transformed into the DAY286 strain of C. albicans. Candida-derived IL-17A was indistinguishable from murine IL-17A in terms of biological activity and detection in standard enzyme-linked immunosorbent assays (ELISAs). Expression of IL-17A did not negatively impact the growth of these strains in vitro. Moreover, the IL-17A-expressing C. albicans strains showed significantly reduced pathogenicity in a systemic model of Candida infection, mainly evident during the early stages of disease. Collectively, these findings suggest that IL-17A mitigates the virulence of C. albicans.

Erratum: Erythropoietin-mediated activation of JAK-STAT signaling contributes to cellular invasion in head and neck squamous cell carcinoma (Oncogene (2005) 24 (4442-4449) DOI:10.1038/sj.onc.1208635)

Correction to: Oncogene 2005; 24: 4442–4449. doi: 10.1038/sj.onc.1208635; published online 18 April 2005 Since publication of the above manuscript, the authors have identified an unintentional omission in the References, and wish to state the following: Another paper describing the expression and invasion promoting ability of erythropoietin (EPO) and its receptor (EPOR) in head and neck cancer cells was recently published: Mohyeldin A, et al. Erythropoietin signaling promotes invasiveness of human head and neck squamous cell carcinoma.