Differential Anti-Tumor Effects of IFN-Inducible Chemokines CXCL9, CXCL10, and CXCL11 on a Mouse Squamous Cell Carcinoma Cell Line.

Chemokines are a group of cytokines involved in the mobilization of leukocytes, which play a role in host defense and a variety of pathological conditions, including cancer. Interferon (IFN)-inducible chemokines C-X-C motif ligand 9 (CXCL), CXCL10, and CXCL11 are anti-tumor chemokines; however, the differential anti-tumor effects of IFN-inducible chemokines are not completely understood. In this study, we investigated the anti-tumor effects of IFN-inducible chemokines by transferring chemokine expression vectors into a mouse squamous cell carcinoma cell line, SCCVII, to generate a cell line stably expressing chemokines and transplanted it into nude mice. The results showed that CXCL9- and CXCL11-expressing cells markedly inhibited tumor growth, whereas CXCL10-expressing cells did not inhibit growth. The NH2-terminal amino acid sequence of mouse CXCL10 contains a cleavage sequence by dipeptidyl peptidase 4 (DPP4), an enzyme that cleaves the peptide chain of chemokines. IHC staining indicated DPP4 expression in the stromal tissue, suggesting CXCL10 inactivation. These results suggest that the anti-tumor effects of IFN-inducible chemokines are affected by the expression of chemokine-cleaving enzymes in tumor tissues.

Enhanced Cytotoxic Effects in Human Oral Squamous Cell Carcinoma Cells Treated with Combined Methyltransferase Inhibitors and Histone Deacetylase Inhibitors.

Combined treatment of human oral squamous cell carcinoma (OSCCs) with DNA methyltransferase inhibitors (DNMTis), histone methyltransferase inhibitors (HMTis), and histone deacetylase inhibitors (HDACis), and the molecular mechanisms underlying their anticancer effects, have not been fully elucidated. Herein, we investigated the cytotoxic effects of combined DNMTis (5-Aza-deoxycytidine: 5-Aza-dC, RG108), HMTis (3-deazaneplanocin A: DZNep), and HDACis (trichostatin A: TSA) treatment on human OSCC cells and explored their molecular mechanisms. Combined 5-Aza-dC, or RG108, and TSA treatment significantly decreased HSC-2 and Ca9-22 cell viability. Combinatorial DZNep and TSA treatment also decreased Ca9-22 cell viability. Although caspase 3/7 activation was not observed in HSC-2 cells following combined treatment, caspase activity was significantly increased in Ca9-22 cells treated with DZNep and TSA. Moreover, combined treatment with 5-Aza-dC, RG108, and TSA increased the proportion of HSC-2 and Ca9-22 cells in the S and G2/M phases. Meanwhile, increased phosphorylation of the histone variant H2A.X, a marker of double-stranded DNA breaks, was observed in both cells after combination treatment. Hence, the decreased viability induced by combined treatment with epigenomic inhibitors results from apoptosis and cell cycle arrest in S and G2/M phases. Thus, epigenomic therapy comprising combined low concentrations of DNMTi, HMTi, and HDACi is effective against OSCC.

Simultaneous Expression of Th1- and Treg-Associated Chemokine Genes and CD4+, CD8+, and Foxp3+ Cells in the Premalignant Lesions of 4NQO-Induced Mouse Tongue Tumorigenesis.

Chemokines and cytokines in the tumor microenvironment influence immune cell infiltration and activation. To elucidate their role in immune cell recruitment during oral cancer development, we generated a mouse tongue cancer model using the carcinogen 4-nitroquinoline 1-oxide (4NQO) and investigated the carcinogenetic process and chemokine/cytokine gene expression kinetics in the mouse tongue. C57/BL6 mice were administered 4NQO in drinking water, after which tongues were dissected at 16 and 28 weeks and subjected to analysis using the RT2 Profiler PCR Array, qRT-PCR, and pathologic and immunohistochemical analyses. We found that Th1-associated chemokine/cytokine (Cxcl9, Cxcl10, Ccl5, and Ifng) and Treg-associated chemokine/cytokine (Ccl17, Ccl22, and Il10) mRNA levels were simultaneously increased in premalignant lesions of 4NQO-treated mice at 16 weeks. Additionally, although levels of Gata3, a Th2 marker, were not upregulated, those of Cxcr3, Ccr4, and Foxp3 were upregulated in the tongue tissue. Furthermore, immunohistochemical analysis confirmed the infiltration of CD4+, CD8+, and Foxp3+ cells in the tongue tissue of 4NQO-treated mice, as well as significant correlations between Th1- or Treg-associated chemokine/cytokine mRNA expression and T cell infiltration. These results indicate that CD4+, CD8+, and Foxp3+ cells were simultaneously recruited through the expression of Th1- and Treg-associated chemokines in premalignant lesions of 4NQO-induced mouse tongue tissue.

Silencing of the interferon-inducible gene Ifi204/p204 induces resistance to interferon-γ-mediated cell growth arrest of tumor cells.

Many tumor cells escape from cancer immunosurveillance and resist treatment with interferons (IFNs). Although the mechanism underlying IFN resistance is mostly attributed to a deficiency of components of the IFN-signaling pathway, some types of tumor cells resist IFN-mediated cell growth arrest despite the presence of an intact JAK/STAT signaling pathway. However, the molecular mechanisms underlying the unresponsiveness to IFNs independent of the defective JAK/STAT pathway remain to be clarified. To elucidate the mechanisms underlying IFNγ resistance, we examined the anti-proliferative effect of IFNγ on mouse tumor cell lines. Mouse squamous cell carcinoma (SCCVII) cells were resistant to IFNγ-mediated cell growth arrest despite the presence of the IFNγ-induced STAT1-dependent signaling pathway, whereas IFNγ inhibited cell growth of B16/F1 cells, a well-known IFNγ-sensitive mouse melanoma cell line, at the G1 phase of the cell cycle. Treatment of SCCVII cells with IFNγ neither downregulated the expression of cyclin D1, cyclin A2, and cyclin E1 nor induced a hypo-phosphorylated, active form of retinoblastoma protein (pRb). Interestingly, the hyper-phosphorylated, inactive form of pRb was exclusively localized in the cytoplasm in SCCVII cells. The IFN-inducible 204 gene (Ifi204), whose gene product, p204, binds to pRb and exerts an anti-proliferative effect, was repressed in SCCVII cells. p204 overexpression in SCCVII significantly inhibited cell growth, and mutation of a pRb-binding LXCXE motif decreased the anti-proliferative effect. These results suggest that silencing of Ifi204/p204 induces resistance to IFNγ-mediated cell growth arrest in SCCVII cells.

Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria.

During chronic inflammation from diseases, such as periodontal disease, the proinflammatory cytokines interferon-gamma (IFNγ) and tumor necrosis factor-α (TNFα) alter bone remodeling. To elucidate the underlying molecular mechanisms, we investigated the effect of IFNγ and TNFα on the proliferation and survival of clonal MC3T3-E1 mouse osteoblasts. We found that although IFNγ or TNFα alone affected cell growth and survival only marginally, costimulation with both synergistically inhibited cell growth and reduced cell viability. The diminished cell viability was due to apoptosis, as indicated by increased TUNEL staining and elevated caspase 3, 8, and 9 activities. Western blot also showed that costimulation with IFNγ and TNFα elicited cytochrome c release and downregulated B cell lymphoma 2 (Bcl-2) expression without affecting Bcl-2-associated X (Bax) protein expression. Furthermore, stable Bcl-2 overexpression significantly alleviated cell death following costimulation. Collectively, these results suggested that IFNγ and TNFα elicited osteoblast apoptosis via cytochrome c release from damaged mitochondria, caspase activation, and Bcl-2 downregulation.

Anti-inflammatory cytokine interleukin-4 inhibits inducible nitric oxide synthase gene expression in the mouse macrophage cell line RAW264.7 through the repression of octamer-dependent transcription.

Inducible nitric oxide synthase (iNOS) is a signature molecule involved in the classical activation of M1 macrophages and is induced by the Nos2 gene upon stimulation with Th1-cell derived interferon-gamma (IFNγ) and bacterial lipopolysaccharide (LPS). Although the anti-inflammatory cytokine IL-4 is known to inhibit Nos2 gene expression, the molecular mechanism involved in the negative regulation of Nos2 by IL-4 remains to be fully elucidated. In the present study, we investigated the mechanism of IL-4-mediated Nos2 transcriptional repression in the mouse macrophage-like cell line RAW264.7. Signal transducer and activator of transcription 6 (Stat6) knockdown by siRNA abolished the IL-4-mediated inhibition of Nos2 induced by IFNγ/LPS. Transient transfection of a luciferase reporter gene containing the 5'-flanking region of the Nos2 gene demonstrated that an octamer transcription factor (OCT) binding site in the promoter region is required for both positive regulation by IFNγ/LPS and negative regulation by IL-4. Although IL-4 had no inhibitory effect on the DNA-binding activity of constitutively expressed Oct-1, IL-4-induced Nos2-reporter transcriptional repression was partially attenuated by overexpression of the coactivator CREB-binding protein (CBP). These results suggest that a coactivator/cofactor that functionally interacts with Oct-1 is a molecular target for the IL-4-mediated inhibition of Nos2 and that IL-4-activated Stat6 represses Oct-1-dependent transcription by competing with this coactivator/cofactor.

Suppression of IP-10/CXCL10 gene expression in LPS- and/or IFN-γ-stimulated macrophages by parasite-secreted products.

T helper (Th)2 polarized immune responses are characteristically dominant in helminth infections. The gene expression of interferon (IFN)-γ-inducible protein 10 (IP-10/CXCL10), which promotes Th1 responses, in mouse macrophages stimulated with lipopolysaccharide (LPS) and/or IFN-γ was suppressed by excretory/secretory (ES) products of Spirometra erinaceieuropaei plerocercoids. ES products suppressed LPS- and/or IFN-γ-induced transcriptional activities of a luciferase reporter gene under the control of a 243-bp fragment of the IP-10 gene promoter/enhancer, which contains an IFN-stimulated response element (ISRE) and two κB elements. Consistent with this result, ES products inhibited ISRE-dependent heterologous promoter activities and LPS- or IFN-γ-induced ISRE-binding activity. ES products also suppressed LPS-induced IFN-ß gene expression. Furthermore, ES products suppressed nuclear factor (NF)-κB RelA (p65)-dependent transcriptional activity, whereas ES products had no effect on the κB-binding activity. These results suggest that ES products suppress the IP-10 gene expression by inhibiting the ISRE- and RelA-dependent transcriptional activities in mouse macrophages.

Infiltration of m2 tumor-associated macrophages in oral squamous cell carcinoma correlates with tumor malignancy.

Tumor-associated macrophages (TAMs) are a major cellular component in the tumor microenvironment of many solid tumors. The functional competence of TAMs varies depending on the type of tumors and their respective microenvironments. The classically activated M1 macrophages exhibit antitumor functions, whereas the alternatively activated M2 macrophages exhibit protumor functions that contribute to tumor development and progression. Although TAMs have been detected in oral squamous cell carcinoma (OSCC), little is known about their phenotype. In the present study, we performed an immunohistochemical analysis to identify TAMs in surgically resected specimens from 50 patients with OSCC and evaluated the relationship between infiltrated TAMs and the pathological grade of OSCC. Positive staining for CD163, which has been used as a marker for M2 macrophages, was observed in OSCC specimens, and the percentages of CD163+ cells were significantly increased based on the pathological grade. CD163+ cells were detected in the tumor stroma in grade I tumors, whereas an increase in the CD163+ cells in the tumor nest was observed in higher grades of tumors. Although infiltrated CD4+ and CD8+ T cells were detected in all pathological grades of OSCC, no correlation between the infiltrated T cells and the CD163+ TAMs was observed. These results indicate that the infiltrated TAMs in OSCC have an M2 phenotype and that the M2 macrophages may participate in the development of OSCC.

Identification of a structural motif in the tumor-suppressive protein GRIM-19 required for its antitumor activity.

We have previously isolated GRIM-19, a novel growth suppressor, using a genetic method. GRIM-19 ablates cell growth by inhibiting the transcription factor signal transducer and activator of transcription 3 (STAT3). Up-regulation of STAT3 and growth promotion were observed in a number of human tumors. Although the tumor-suppressive actions of GRIM-19 are known, the structural elements required for its antitumor actions are not understood. Mutational and protein sequence analyses identified a motif in the N terminus of GRIM-19 that exhibited similarity to certain RNA viral proteins. We show that disruption of specific amino acids within this motif cripples the antitumor actions of GRIM-19. These mutants fail to interact with STAT3 efficiently and consequently do not inhibit growth-promoting gene expression. More importantly, we show that a clinically observed mutation in the N terminus of GRIM-19 also weakened its interaction with STAT3 and antitumor action. Together, these studies identify a major role for the N terminus of GRIM-19 in mediating its tumor-suppressive actions.

STAT1 represses hypoxia-inducible factor-1-mediated transcription.

Tumor hypoxia is associated with tumor promotion, malignant progression, and resistance to cancer therapy. The hypoxia-induced phenotypic changes in tumors result, at least partially, from the induction of hypoxia-responsive genes, such as chemokine receptor CXCR4. Hypoxia-inducible factor 1 (HIF-1) is a critical transcription factor involved in the transcriptional regulation of these genes. Although interferon-gamma (IFNgamma) exerts anti-tumor responses against various tumors, the effect of IFNgamma on HIF-1-dependent transcription remains to be determined. In this study, we examined the inhibitory effect of IFNgamma on hypoxia-induced CXCR4 gene expression in human glioblastoma cell lines and explored the mechanism of inhibition. Hypoxia (1% O(2)) and the iron chelator deferoxamine (DFX), a hypoxia mimetic, increased the levels of CXCR4 mRNA in A172 and T98G cells, and treatment with IFNgamma inhibited the expression of CXCR4 mRNA. Although hypoxia and DFX induced the expression of HIF-1alpha protein and its hypoxia response element (HRE) DNA-binding activity, IFNgamma failed to inhibit its expression or DNA-binding activity. The results of a luciferase reporter assay using a heterologous promoter construct containing the HRE sequence revealed that IFNgamma suppressed the hypoxia- and DFX-induced reporter activities. Lentivirus-mediated RNAi of signal transducer and activator of transcription 1 (STAT1) expression abolished the inhibitory effect of IFNgamma on hypoxia-induced reporter gene activity. Furthermore, this activity was not detected in a stable cell line expressing dominant-negative STAT1. These results indicate that IFNgamma-activated STAT1 functions as a negative regulator of HIF-1-dependent transcription in tumor cells.

Mechanisms of resistance to interferon-gamma-mediated cell growth arrest in human oral squamous carcinoma cells.

Interferon-gamma (IFNgamma) has an antiproliferative effect on a variety of tumor cells. However, many tumor cells resist treatment with IFNs. Here, we show that IFNgamma fails to inhibit the growth of some types of oral squamous cell carcinoma (OSCC) cells that possess a fully functional IFNgamma/STAT1 (signal transducer and activator of transcription-1) signaling pathway. IFNgamma inhibited the growth of the HSC-2, HSC-3, and HSC-4 OSCC cell lines. However, Ca9-22 cells were resistant to IFNgamma despite having intact STAT1-dependent signaling, such as normal tyrosine phosphorylation, DNA binding activity, and transcriptional activity of STAT1. The growth inhibition of HSC-2 cells resulted from S-phase arrest of the cell cycle. IFNgamma inhibited cyclin A2 (CcnA2)-associated kinase activity, which correlated with the IFNgamma-mediated down-regulation of CcnA2 and Cdk2 expression at both the transcriptional and post-transcriptional level in HSC-2 cells but not in Ca9-22 cells. RNAi-mediated knockdown of CcnA2 and Cdk2 resulted in growth inhibition in both cell lines. These results indicate that the resistance of OSCC to IFNgamma is not due simply to the deficiency in STAT1-dependent signaling but results from a defect in the signaling component that mediates this IFNgamma-induced down-regulation of CcnA2 and Cdk2 expression at the transcriptional and post-transcriptional levels.

Loss of CYLD might be associated with development of salivary gland tumors.

Molecular studies of cylindromas, which arise from the eccrine or apocrine cells of the skin, have demonstrated frequent alterations at chromosome 16q12-13, recently found to house the cylindromatosis (CYLD) gene. CYLD, a tumor suppressor gene, has deubiquitinating enzyme activity and inhibits the activation of transcription factor NF-kappaB. Loss of the deubiquitinating activity of CYLD is correlated with tumorigenesis. It has been reported that the expression of CYLD is observed in various organs. We demonstrated previously that human salivary gland tumor (SGT) cell line, HSG spontaneously expresses CYLD and also found that adenoid cystic carcinoma (ACC) arising from the hard palate was distinctly positive for CYLD, immunohistochemically. However, it is unclear whether loss of CYLD is associated with development of SGTs. This study examined CYLD function in SGT cells and attempted to clarify whether CYLD is associated with development of SGTs. The expression of CYLD and NF-kappaB mRNAs in HSG cells was increased by TNF-alpha. Translocation of NF-kappaB protein from the cytoplasm to the nucleus in HSG cells peaked at 30 min after TNF-alpha stimulation, then decreased at 60 min, whereas that of CYLD protein increased gradually in a time-dependent manner. Luciferase reporter assay indicated that TNF-alpha induced a 5-fold increase of NF-kappaB-dependent transcription at 4 h, which was further enhanced by knockdown of CYLD using RNA interference. Taken together, these data demonstrated that the levels of both CYLD and NF-kappaB mRNAs accumulated in HSG cells during 24 h after TNF-alpha stimulation, although the NF-kappaB activity in the cells was at least negatively regulated by CYLD. Immunohistochemical examinations revealed that there are several correlations between the expression of CYLD and NF-kappaB-related factors in 17 cases of ACC tissues. These findings suggest that loss of CYLD is associated with development of SGTs.

Influenza A virus abrogates IFN-gamma response in respiratory epithelial cells by disruption of the Jak/Stat pathway.

The innate immunity to viral infections induces a potent antiviral response mediated by interferons (IFN). Although IFN-gamma is detected during the acute stages of illness in the upper respiratory tract secretions and in the serum of influenza A virus-infected individuals, control of influenza A virus is not dependent upon IFN-gamma as evidenced by studies using anti-IFN-gamma Ab and IFN-gamma(-/-) mice. Thus, we hypothesized that IFN-gamma is not critical in host survival because influenza A virus has mechanisms to evade the antiviral activity of IFN-gamma. To test this, A549 cells, an epithelial cell line derived from lung adenocarcinoma, were infected with influenza virus strain A/Aichi/2/68 (H3N2) (Aichi) and/or stimulated with IFN-gamma to detect IFN-gamma-stimulated MHC class II expression. Influenza A virus infection inhibited IFN-gamma-induced up-regulation of HLA-DRalpha mRNA and the IFN-gamma induction of class II transactivator (CIITA), an obligate mediator of MHC class II expression. Nuclear translocation of Stat1alpha upon IFN-gamma stimulation was significantly inhibited in influenza A virus-infected cells and this was associated with a decrease in Tyr701 and Ser727 phosphorylation of Stat1alpha. Thus, influenza A virus subverts antiviral host defense mediated by IFN-gamma through effects on the intracellular signaling pathways.

Sulindac, a nonsteroidal anti-inflammatory drug, selectively inhibits interferon-gamma-induced expression of the chemokine CXCL9 gene in mouse macrophages.

Sulindac, a non-steroidal anti-inflammatory drug, has been shown to exert an anti-tumor effect on several types of cancer. To determine the effect of sulindac on intracellular signaling pathways in host immune cells such as macrophages, we investigated the effect of the drug on interferon gamma (IFNgamma)-induced expression of signal transducer and activator of transcription 1 (STAT1) and other genes in mouse macrophage-like cell line RAW264.7 cells. Sulindac, but not aspirin or sodium salicylate, inhibited IFNgamma-induced expression of the CXC ligand 9 (CXCL9) mRNA, a chemokine for activated T cells, whereas the interferon-induced expression of CXCL10 or IFN regulatory factor-1 was not affected by sulindac. Luciferase reporter assay demonstrated that sulindac inhibited IFNgamma-induced promoter activity of the CXCL9 gene. Surprisingly, sulindac had no inhibitory effect on IFNgamma-induced STAT1 activation; however, constitutive nuclear factor kappaB activity was suppressed by the drug. These results indicate that sulindac selectively inhibited IFNgamma-inducible gene expression without inhibiting STAT1 activation.

Constitutive nuclear factor kappaB activity is required to elicit interferon-gamma-induced expression of chemokine CXC ligand 9 (CXCL9) and CXCL10 in human tumour cell lines.

CXC ligand 10 (CXCL10) and CXCL9 are chemoattractants for activated T cells and possess angiostatic activity. Both CXCL9 and CXCL10 have been considered as important components for the anti-tumour activities of interferon-gamma (IFNgamma) and interleukin-12 in animal models. In this article we show that the CXCL9 and CXCL10 genes in some types of human tumour cell lines are not inducible by IFNgamma and we describe experiments designed to explore the molecular mechanisms involved in this impaired induction. The human oral squamous carcinoma line Ca9-22 and the glioma line A172 failed to express CXCL9 and CXCL10 mRNAs in response to IFNgamma, whereas other carcinoma lines including HSC-2 did express these mRNAs. Production of these chemokine proteins was also impaired in Ca9-22 cells. The impaired expression was not due to any deficiency in the IFNgamma/signal transducer and activator of transcription 1 (STAT1)-dependent signalling pathway. Instead, analysis of nuclear factor kappaB (NF-kappaB) activity revealed that the constitutive low level of NF-kappaB activity, which is seen in cells that express these chemokines, was absent in Ca9-22 and A172 cells. Activation of NF-kappaB in Ca9-22 cells restored the expression of IFNgamma-stimulated CXCL9 and CXCL10 mRNAs. In contrast, inhibition of the constitutive NF-kappaB in HSC-2 cells by adenovirus-mediated gene transfer of a dominant-negative IkappaBalpha suppressed the IFNgamma-induced expression of the CXCL9 and CXCL10 mRNAs. These results indicate that constitutive NF-kappaB activity, which is often associated with tumour development, is required for the induced expression of CXCL9 and CXCL10 genes in human tumour cell lines in response to IFNgamma.

The transcriptional coactivator CREB-binding protein cooperates with STAT1 and NF-kappa B for synergistic transcriptional activation of the CXC ligand 9/monokine induced by interferon-gamma gene.

Signal transducers and activators of transcription 1 (STAT1) and NF-kappaB cooperatively regulate the expression of many inflammatory genes. In the present study, we demonstrate that the transcriptional coactivator CREB-binding protein (CBP) mediated the STAT1/NF-kappaB synergy for transcription of the gene for CXC ligand 9 (CXCL9), an interferon-gamma (IFN-gamma)-inducible chemokine. Reporter gene analysis showed that expression of CBP potentiated IFN-gamma and tumor necrosis factor (TNFalpha)-induced promoter activity and that the CBP-mediated synergy depended upon STAT1- and NF-kappaB-binding sites in the promoter. Experiments with CBP mutants indicated that the N-terminal and C-terminal regions were necessary for the transcriptional synergy, although the histone acetyltransferase activity of CBP was dispensable. A co-immunoprecipitation assay demonstrated that STAT1 and NF-kappaB RelA (p65) simultaneously associated with CBP in vivo. Furthermore, chromatin immunoprecipitation revealed that, although costimulation with IFN-gamma and TNFalpha did not cooperatively enhance the levels of acetylated histones, it did result in increased recruitment of STAT1, CBP, and RNA polymerase II at the promoter region of the CXCL 9 gene. Together, these results demonstrate that the STAT1/NF-kappaB-dependent transcriptional synergy could result from the enhanced recruitment of RNA polymerase II complex to the promoter via simultaneous interaction of CBP with STAT1 and NF-kappaB.