Research IndicatorsGraph generated 27 February 2015 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex
Specific Cancers (2)
Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.
Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
Search the Epigenomics database and view relevant gene tracks of samples.
Latest Publications: IRF7 (cancer-related)
Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA - Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints - in particular the PD-1/PD-L1 pathway - may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade.
Ettahar A, Ferrigno O, Zhang MZ, et al.Identification of PHRF1 as a tumor suppressor that promotes the TGF-β cytostatic program through selective release of TGIF-driven PML inactivation.
Cell Rep. 2013; 4(3):530-41 [PubMed
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The homeodomain protein TGIF (TG-interacting factor) restricts TGF-β/Smad cytostatic signaling by interfering with the nucleocytoplasmic transit of the tumor suppressor cPML. Here, we identify PHRF1 as a ubiquitin ligase that enforces TGIF decay by driving its ubiquitination at lysine 130. In so doing, PHRF1 ensures redistribution of cPML into the cytoplasm, where it associates with SARA and coordinates activation of Smad2 by the TGF-β receptor. The PHRF1 gene resides within the tumor suppressor locus 11p15.5, which displays frequent loss in a wide variety of malignancies, including breast cancer. Remarkably, we found that the PHRF1 gene is deleted or silenced in a high proportion of human breast cancer samples and cancer cell lines. Reconstitution of PHRF1 into deficient cells impeded their propensity to form tumors in vivo, most likely because of the reemergence of TGF-β responsiveness. These findings unveil a paradigm behind inactivation of the cPML tumor suppressor network in human malignancies.
The principal Epstein-Barr virus (EBV) oncoprotein, Latent Membrane Protein 1 (LMP1), is expressed in most EBV-associated human malignancies. LMP1 mimics CD40 receptor signaling to provide infected cells with constitutive NF-κB, MAP kinase, IRF7, and PI3 kinase pathway stimulation. EBV-transformed B-cells are particularly dependent on constitutive NF-κB activity, and rapidly undergo apoptosis upon NF-κB blockade. Here, we review LMP1 function, with special attention to current understanding of the molecular mechanisms of LMP1-mediated NF-κB and IRF7 pathway activation. Recent advances include the elucidation of transmembrane motifs important for LMP1 trafficking and ligand-independent signaling, analysis of genome-wide LMP1 gene targets, and the identification of novel cell proteins that mediate LMP1 NF-κB and IRF7 pathway activation.
Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) induces multiple signal transduction pathways during latent EBV infection via its C-terminal activating region 1 (CTAR1), CTAR2, and the less-studied CTAR3. One mechanism by which LMP1 regulates cellular activation is through the induction of protein posttranslational modifications, including phosphorylation and ubiquitination. We recently documented that LMP1 induces a third major protein modification by physically interacting with the SUMO-conjugating enzyme Ubc9 through CTAR3 and inducing the sumoylation of cellular proteins in latently infected cells. We have now identified a specific target of LMP1-induced sumoylation, interferon regulatory factor 7 (IRF7). We hypothesize that during EBV latency, LMP1 induces the sumoylation of IRF7, limiting its transcriptional activity and modulating the activation of innate immune responses. Our data show that endogenously sumoylated IRF7 is detected in latently infected EBV lymphoblastoid cell lines. LMP1 expression coincided with increased sumoylation of IRF7 in a CTAR3-dependent manner. Additional experiments show that LMP1 CTAR3-induced sumoylation regulates the expression and function of IRF7 by decreasing its turnover, increasing its nuclear retention, decreasing its DNA binding, and limiting its transcriptional activation. Finally, we identified that IRF7 is sumoylated at lysine 452. These data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling, leading to biologic effects. We propose that CTAR3 is an important signaling region of LMP1 that regulates protein function by sumoylation. We have shown specifically that LMP1 CTAR3, in cooperation with CTAR2, can limit the ability of IRF7 to induce innate immune responses by inducing the sumoylation of IRF7.
Bidwell BN, Slaney CY, Withana NP, et al.Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.
Nat Med. 2012; 18(8):1224-31 [PubMed
] Related Publications
Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8(+) T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis-free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.
Knowledge of oncogenic mutations can inspire therapeutic strategies that are synthetically lethal, affecting cancer cells while sparing normal cells. Lenalidomide is an active agent in the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), but its mechanism of action is unknown. Lenalidomide kills ABC DLBCL cells by augmenting interferon β (IFNβ) production, owing to the oncogenic MYD88 mutations in these lymphomas. In a cereblon-dependent fashion, lenalidomide downregulates IRF4 and SPIB, transcription factors that together prevent IFNβ production by repressing IRF7 and amplify prosurvival NF-κB signaling by transactivating CARD11. Blockade of B cell receptor signaling using the BTK inhibitor ibrutinib also downregulates IRF4 and consequently synergizes with lenalidomide in killing ABC DLBCLs, suggesting attractive therapeutic strategies.
BACKGROUND: Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined.
PRINCIPAL FINDINGS: In this study, we demonstrate that virus infection of Namalwa cells induces transient recruitment of HDAC3 (histone deacetylase 3) to IFN-A promoters. Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription. Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression. Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription.
CONCLUSION: Altogether these data indicate that reversal of histone H3K9/K14 acetylation by HDAC3 is required for attenuation of IFN-A gene transcription during viral infection.
Moschonas A, Ioannou M, Eliopoulos AGCD40 stimulates a "feed-forward" NF-κB-driven molecular pathway that regulates IFN-β expression in carcinoma cells.
J Immunol. 2012; 188(11):5521-7 [PubMed
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IFN-β and the CD40L (CD154) share important roles in the antiviral and antitumor immune responses. In this study, we show that CD40 receptor occupancy results in IFN-β upregulation through an unconventional "feed-forward" mechanism, which is orchestrated by canonical NF-κB and involves the sequential de novo synthesis of IFN regulatory factor (IRF)1 and Viperin (RSAD2), an IRF1 target. RelA (p65) NF-κB, IRF1, and Viperin-dependent IRF7 binding to the IFN-β promoter largely controls its activity. However, full activation of IFN-β also requires the parallel engagement of noncanonical NF-κB2 signaling leading to p52 recruitment to the IFN-β promoter. These data define a novel link between CD40 signaling and IFN-β expression and provide a telling example of how signal propagation can be exploited to ensure efficient regulation of gene expression.
BACKGROUND: The cysteinyl leukotrienes receptors (CysLTRs) are implicated in many different pathological conditions, such as inflammation and cancer. We have previously shown that colon cancer patients with high CysLT(1)R and low CysLT(2)R expression demonstrate poor prognosis. Therefore, we wanted to investigate ways for the transcriptional regulation of CysLT(2)R, which still remains to be poorly understood.
METHODOLOGY/PRINCIPAL FINDINGS: We investigated the potential role of the anti-tumorigenic interferon α (IFN-α) and the mitogenic epidermal growth factor (EGF) on CysLT(2)R regulation using non-transformed intestinal epithelial cell lines and colon cancer cells to elucidate the effects on the CysLT(2)R expression and regulation. This was done using Western blot, qPCR, luciferase reporter assay and a colon cancer patient array. We found a binding site for the transcription factor IRF-7 in the putative promoter region of CysLT(2)R. This site was involved in the IFN-α induced activity of the CysLT(2)R luciferase reporter assay. In addition, IFN-α induced the activity of the differentiation marker alkaline phosphatase along with the expression of mucin-2, which protects the epithelial layer from damage. Interestingly, EGF suppressed both the expression and promoter activity of the CysLT(2)R. E-boxes present in the CysLT(2)R putative promoter region were involved in the suppressing effect. CysLT(2)R signaling was able to suppress cell migration that was induced by EGF signaling.
CONCLUSIONS/SIGNIFICANCE: The patient array showed that aggressive tumors generally expressed less IFN-α receptor and more EGFR. Interestingly, there was a negative correlation between CysLT(2)R and EGFR expression. Our data strengthens the idea that there is a protective role against tumor progression for CysLT(2)R and that it highlights new possibilities to regulate the CysLT(2)R.
Defective IFN signaling results in loss of innate immunity and sensitizes cells to enhanced cytolytic killing after Vesticular Stomatitis Virus (VSV) infection. Examination of the innate immunity status of normal human bronchial epithelial cells Beas2B and 7 lung cancer cells revealed that the abrogation of IFN signaling in cancer cells is associated with greater sensitivity to VSV infection. The disruption of the IFN pathway in lung cancer cell lines and primary tumor tissues is caused by epigenetic silencing of critical interferon responsive transcription factors IRF7 and/or IRF5. Although 5-aza-2'-deoxycytidine treatment fails to reactivate IRF7 and IRF5 expression or protect cells from VSV infection, manipulating IFN signaling by altering IRF expression changes the viral susceptibility of these cells. Lung cancer cells can be partially protected from viral killing using IRF5+IRF7 overexpression, whereas IFN pathway disruption by transfection of siRNAs to IRF5+IRF7 increases cells' vulnerability to viral infection. Therefore, IRF5 and IRF7 are key transcription factors in IFN pathway that determine viral sensitivity of lung cancer cells; the epigenetically impaired IFN pathway in lung cancer tissues provides potential biomarkers for successful selective killing of cancer cells by oncolytic viral therapy.
Lee J, Li L, Gretz N, et al.Absent in Melanoma 2 (AIM2) is an important mediator of interferon-dependent and -independent HLA-DRA and HLA-DRB gene expression in colorectal cancers.
Oncogene. 2012; 31(10):1242-53 [PubMed
] Free Access to Full Article Related Publications
Absent in Melanoma 2 (AIM2) is a member of the HIN-200 family of hematopoietic, IFN-inducible, nuclear proteins, associated with both, infection defense and tumor pathology. Recently, AIM2 was found to act as a DNA sensor in innate immunity. In addition, we and others have previously demonstrated a high frequency of AIM2-alterations in microsatellite unstable (MSI-H) tumors. To further elucidate AIM2 function in colorectal tumors, we here addressed AIM2-responsive target genes by microarray based gene expression profiling of 22 244 human genes. A total of 111 transcripts were significantly upregulated, whereas 80 transcripts turned out to be significantly downregulated in HCT116 cells, constitutively expressing AIM2, compared with AIM2-negative cells. Among the upregulated genes that were validated by quantitative PCR and western blotting we recognized several interferon-stimulated genes (ISGs: IFIT1, IFIT2, IFIT3, IFI6, IRF7, ISG15, HLA-DRA, HLA-DRB, TLR3 and CIITA), as well as genes involved in intercellular adhesion and matrix remodeling. Expression of ISGs correlated with expression of AIM2 in 10 different IFN-γ treated colorectal cancer cell lines. Moreover, small interfering RNA-mediated knock-down of AIM2 resulted in reduced expression of HLA-DRA, HLA-DRB and CIITA in IFN-γ-treated cells. IFN-γ independent induction of HLA-DR genes and their encoded proteins was also demonstrated upon doxycyclin-regulated transient induction of AIM2. Luciferase reporter assays revealed induction of the HLA-DR promoter upon AIM2 transfection in different cell lines. STAT-signaling was not involved in IFN-γ independent induction of ISGs, arguing against participation of cytokines released in an autostimulating manner. Our data indicate that AIM2 mediates both IFN-γ dependent and independent induction of several ISGs, including genes encoding the major histocompatibility complex (MHC) class II antigens HLA-DR-α and -β. This suggests a novel role of the IFN/AIM2/ISG cascade likewise in cancer cells.
Wang L, Toomey NL, Diaz LA, et al.Oncogenic IRFs provide a survival advantage for Epstein-Barr virus- or human T-cell leukemia virus type 1-transformed cells through induction of BIC expression.
J Virol. 2011; 85(16):8328-37 [PubMed
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miR-155, processed from the B-cell integration cluster (BIC), is one of the few well-studied microRNAs (miRNAs) and is involved in both innate immunity and tumorigenesis. BIC/miR-155 is induced by distinct signaling pathways, but little is known about the underlying mechanisms. We have identified two conserved potential interferon (IFN) regulatory factor (IRF)-binding/interferon-stimulated response element motifs in the Bic gene promoter. Two oncogenic IRFs, IRF4 and -7, in addition to some other members of the family, bind to and significantly transactivate the Bic promoter. Correspondingly, the endogenous levels of IRF4 and -7 are correlated with that of the BIC transcript in Epstein-Barr virus (EBV)-transformed cells. However, RNA interference studies have shown that depletion of IRF4, rather than of IRF7, dramatically decreases the endogenous level of BIC by up to 70% in EBV- or human T-cell leukemia virus type 1 (HTLV1)-transformed cell lines and results in apoptosis and reduction of proliferation rates that are restored by transient expression of miR-155. Moreover, the endogenous levels of the miR-155 target, SHIP1, are consistently elevated in EBV- and HTLV1-transformed cell lines stably expressing shIRF4. In contrast, transient expression of IRF4 decreases the SHIP1 level in EBV-negative B cells. Furthermore, the level of IRF4 mRNA is significantly correlated with that of BIC in adult T-cell lymphoma/leukemia (ATLL) tumors. These results show that IRF4 plays an important role in the regulation of BIC in the context of EBV and HTLV1 infection. Our findings have identified Bic as the first miRNA-encoding gene for IRFs and provide evidence for a novel molecular mechanism underlying the IRF/BIC pathway in viral oncogenesis.
Ning S, Pagano JS, Barber GNIRF7: activation, regulation, modification and function.
Genes Immun. 2011; 12(6):399-414 [PubMed
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Interferon regulatory factor 7 (IRF7) was originally identified in the context of Epstein-Barr virus (EBV) infection, and has since emerged as the crucial regulator of type I interferons (IFNs) against pathogenic infections, which activate IRF7 by triggering signaling cascades from pathogen recognition receptors (PRRs) that recognize pathogenic nucleic acids. Moreover, IRF7 is a multifunctional transcription factor, underscored by the fact that it is associated with EBV latency, in which IRF7 is induced as well as activated by the EBV principal oncoprotein latent membrane protein-1 (LMP1). Aberrant production of type I IFNs is associated with many types of diseases such as cancers and autoimmune disorders. Thus, tight regulation of IRF7 expression and activity is imperative in dictating appropriate type I IFN production for normal IFN-mediated physiological functions. Posttranslational modifications have important roles in regulation of IRF7 activity, exemplified by phosphorylation, which is indicative of its activation. Furthermore, mounting evidence has shed light on the importance of regulatory ubiquitination in activation of IRF7. Albeit these exciting findings have been made in the past decade since its discovery, many questions related to IRF7 remain to be addressed.
BACKGROUND: The expression of microRNAs (miRNAs) is deregulated in acute myeloid leukemia (AML), but the corresponding functional miRNA-controlled pathways are poorly understood. Integration of messenger RNA (mRNA) and miRNA expression profiling may allow the identification of functional links between the whole transcriptome and microRNome that are involved in myeloid leukemogenesis.
METHODS: We integrated miRNA and mRNA expression profiles obtained from 48 newly diagnosed AML patients by using 2 different microarray platforms and performed correlation, gene ontology, and network analysis. Experimental validation was also performed in AML cell lines using miRNA oligonucleotide mimics and functional assays.
RESULTS: Our analysis identified a strong positive correlation between HOX-related genes and miR-10 and miR-20a. Furthermore, we observed a negative correlation between miR-181a and miR-181b, miR-155, and miR-146 expression with that of genes involved in immunity and inflammation (eg, IRF7 and TLR4) and a positive correlation between miR-23a, miR-26a, miR-128a, and miR-145 expression with that of proapoptotic genes (eg, BIM and PTEN). These correlations were confirmed by gene ontology analyses, which revealed the enrichment of members of the homeobox, immunity and inflammation, and apoptosis biological processes. Furthermore, we validated experimentally the association of miR-145, miR-26a, and miR-128a with apoptosis in AML.
CONCLUSION: Our results indicate that by integrating the transcriptome and microRNome in AML cells, it is possible to identify previously unidentified putative functional miRNA-mRNA interactions in AML.
Type I interferon (IFN) signaling is the principal response mediating antiviral innate immunity. IFN transcription is dependent upon the activation of transcription factors IRF3/IRF7 and NF-κB. Many viral proteins have been shown as being capable of interfering with IFN signaling to facilitate evasion from the host innate immune response. Here, we report that a viral miRNA, miR-K12-11, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for the modulation of IFN signaling and acts through targeting I-kappa-B kinase epsilon (IKKɛ). Ectopic expression of miR-K12-11 resulted in decreased IKKɛ expression, while inhibition of miR-K12-11 was found to restore IKKɛ expression in KSHV-infected cells. Importantly, expression of miR-K12-11 attenuated IFN signaling by decreasing IKKɛ-mediated IRF3/IRF7 phosphorylation and by inhibiting the activation of IKKɛ-dependent IFN stimulating genes (ISGs), allowing miR-K12-11 suppression of antiviral immunity. Our data suggest that IKKɛ targeting by miR-K12-11 is an important strategy utilized by KSHV to modulate IFN signaling during the KSHV lifecycle, especially in latency. We also demonstrated that IKKɛ was able to enhance KSHV reactivation synergistically with the treatment of 12-O-tetradecanoylphorbol 13-acetate. Moreover, inhibition of miR-K12-11 enhanced KSHV reactivation induced by vesicular stomatitis virus infection. Taken together, our findings also suggest that miR-K12-11 can contribute to maintenance of KSHV latency by targeting IKKɛ.
The IFN pathway is abrogated in fibroblasts from Li-Fraumeni syndrome (LFS) patients during spontaneous cellular immortalization, a necessary step in carcinogenesis. Microarray profiling of differentially expressed microRNAs (miRNA) revealed that most miRNAs were upregulated in IFN pathway-defective MDAH087-10 fibroblasts compared with MDAH087-N cells with relatively normal IFN signaling. Overexpression of Dicer, a critical enzyme in miRNA biogenesis, promoted cell growth and colony formation in MDAH087-10 cells. However, double-stranded miRNA produced by Dicer enhanced the expression of IFN-stimulated genes in MDAH087-N cells resulting in significant cell death and reduced cell growth. Furthermore, manipulation of the IFN pathway in immortal LFS fibroblasts through transcription factor IRF7 reversed their response to Dicer overexpression due to changed IFN pathway activity. Dicer overexpressing MDAH087-N cells contained lower levels of miRNA than vector control, and conversely much higher miRNA expression was detected in Dicer-transfected MDAH087-10 cells. Therefore, cells with a defective IFN pathway have a higher miRNA tolerance than cells with normal IFN pathway. This work indicates for the first time that the IFN pathway as mediated through the transcription factor IRF7 must be disrupted to permit miRNA upregulation to occur in early carcinogenesis. The IFN pathway appears to provide a checkpoint for miRNA level tolerance and its abrogation leads to cellular immortalization.
Virus infection of a cell generally evokes an immune response by the host to defeat the intruder in its effort. Many viruses have developed an array of strategies to evade or antagonize host antiviral responses. Kaposi's sarcoma-associated herpesvirus (KSHV) is demonstrated in this report to be able to prevent activation of host antiviral defense mechanisms upon infection. Cells infected with wild-type KSHV were permissive for superinfection with vesicular stomatitis virus (VSV), suggesting that KSHV virions fail to induce host antiviral responses. We previously showed that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu et al., Proc. Natl. Acad. Sci. USA. 99:5573-5578, 2002). Here, using an ORF45-null recombinant virus, we demonstrate a profound role of ORF45 in inhibiting host antiviral responses. Infection of cells with an ORF45-null mutant recombinant KSHV (BAC-stop45) triggered an immune response that resisted VSV super-infection, concomitantly associated with appreciable increases in transcription of type I IFN and downstream anti-viral effector genes. Gain-of-function analysis showed that ectopic expression of ORF45 in human fibroblast cells by a lentivirus vector decreased the antiviral responses of the cells. shRNA-mediated silencing of IRF-7, that predominantly regulates both the early and late phase induction of type I IFNs, clearly indicated its critical contribution to the innate antiviral responses generated against incoming KSHV particles. Thus ORF45 through its targeting of the crucial IRF-7 regulated type I IFN antiviral responses significantly contributes to the KSHV survival immediately following a primary infection allowing for progression onto subsequent stages in its life-cycle.
BACKGROUND: FUS1/TUSC2 is a novel tumor suppressor located in the critical 3p21.3 chromosomal region frequently deleted in multiple cancers. We previously showed that Tusc2-deficient mice display a complex immuno-inflammatory phenotype with a predisposition to cancer. The goal of this study was to analyze possible involvement of TUSC2 in malignant pleural mesothelioma (MPM) - an aggressive inflammatory cancer associated with exposure to asbestos.
METHODS: TUSC2 insufficiency in clinical specimens of MPM was assessed via RT-PCR (mRNA level), Representational Oligonucleotide Microarray Analysis (DNA level), and immunohistochemical evaluation (protein level). A possible link between TUSC2 expression and exposure to asbestos was studied using asbestos-treated mesothelial cells and ROS (reactive oxygen species) scavengers. Transcripional effects of TUSC2 in MPM were assessed through expression array analysis of TUSC2-transfected MPM cells.
RESULTS: Expression of TUSC2 was downregulated in approximately 84% of MM specimens while loss of TUSC2-containing 3p21.3 region observed in approximately 36% of MPMs including stage 1 tumors. Exposure to asbestos led to a transcriptional suppression of TUSC2, which we found to be ROS-dependent. Expression array studies showed that TUSC2 activates transcription of multiple genes with tumor suppressor properties and down-regulates pro-tumorigenic genes, thus supporting its role as a tumor suppressor. In agreement with our knockout model, TUSC2 up-regulated IL-15 and also modulated more than 40 other genes (approximately 20% of total TUSC2-affected genes) associated with immune system. Among these genes, we identified CD24 and CD274, key immunoreceptors that regulate immunogenic T and B cells and play important roles in systemic autoimmune diseases. Finally, clinical significance of TUSC2 transcriptional effects was validated on the expression array data produced previously on clinical specimens of MPM. In this analysis, 42 TUSC2 targets proved to be concordantly modulated in MM serving as disease discriminators.
CONCLUSION: Our data support immuno-therapeutic potential of TUSC2, define its targets, and underscore its importance as a transcriptional stimulator of anti-tumorigenic pathways.
We earlier reported that PU.1 was downregulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were downregulated and p21 was upregulated, although among apoptosis-related genes, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was found highly upregulated. When TRAIL was knocked down by small interference RNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL has a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Upregulation of PU.1-induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL.
Karaczyn AA, Cheng RY, Buzard GS, et al.Truncation of histone H2A's C-terminal tail, as is typical for Ni(II)-assisted specific peptide bond hydrolysis, has gene expression altering effects.
Ann Clin Lab Sci. 2009; 39(3):251-62 [PubMed
] Free Access to Full Article Related Publications
Nickel(II), capable of transforming cells and causing tumors in humans and animals, has been previously shown by us to mediate hydrolytic truncation of histone H2A's C-terminal tail by 8 amino acids in both cell-free and cell culture systems. Since H2A's C-tail is involved in maintaining chromatin structure, such truncation might alter this structure and affect gene expression. To test the latter possibility, we transfected cultured T-REx 293 human embryonic kidney cells with plasmids expressing either wild type (wt) or truncated (q) histone H2A proteins, which were either untagged or N-terminally tagged with fluorescent proteins. Each histone variant was found to be incorporated into chromatin at 24 and 48 hr post-transfection. Cells transfected with the untagged plasmids were tested for gene expression by microarray and real-time PCR. Evaluation of the results for over 21,000 genes using the multidimensional scaling and hierarchical clustering methods revealed significant differences in expression of numerous genes between the q-H2A and wt-H2A transfectants. Many of the differentially expressed genes, including BAZ2A, CLDN18, CYP51A1, GFR, GIPC2, HMGB1, IRF7, JAK3, PSIP1, and VEGF, are cancer-related genes. The results thus demonstrate the potential of q-H2A to contribute to the process of carcinogenesis through epigenetic mechanisms.
Jee CD, Kim MA, Jung EJ, et al.Identification of genes epigenetically silenced by CpG methylation in human gastric carcinoma.
Eur J Cancer. 2009; 45(7):1282-93 [PubMed
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To identify novel methylation-silenced genes in gastric cancer, we carried out a genome-wide search for genes that are up-regulated after treatment with the demethylating agent, 5-aza-2'-deoxycytidine (5Aza-dC). When three gastric cancer cell lines (SNU-1,-601, and -719) were treated with 5Aza-dC, 143 genes were found to be upregulated by twofold or more using oligonucleotide microarrays. Six of these genes, i.e. TFPI2, GPX3, GPX1, IGFBP6, IRF7 and DMRT1, showed promoter hypermethylation in one or more gastric cancer cell lines, but were unmethylated in normal gastric mucosa by bisulphite sequencing and methylation-specific PCR analysis. The following percentages of these genes were found to be aberrantly methylated in gastric cancer samples; TFPI2 (80.9%), GPX3 (30.1%), DMRT1 (46.9%), GPX1 (16.7%), IGFBP6 (22.6%) and IRF7 (32.1%). Interestingly, the survival of patients possessing methylated alleles of TFPI2 (123/152, 80.9%) was poorer than that of patients with unmethylated alleles (p=0.023). Multivariate analysis confirmed that TFPI2 methylation is a significant and independent prognostic factor in gastric carcinoma. Furthermore, altered TFPI2 expression, as demonstrated by immunohistochemistry in 566 consecutive gastric cancer tissues, was found to be significantly associated with sex (p=0.003), WHO classification (p<0.001), and a mixed subtype by Lauren's classification (p<0.001). Thus, the present study identified several novel genes, which were methylated in gastric cancer and among them, methylation of TFPI2 was an unfavourable prognostic marker.
Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPalpha, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPalpha was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPalpha was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPalpha markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPalpha and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPalpha localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPalpha causes dysregulation of its function, suggesting that C/EBPalpha is a novel candidate tumor suppressor gene in pancreatic cancer cells.
Liu S, Ren S, Howell P, et al.Identification of novel epigenetically modified genes in human melanoma via promoter methylation gene profiling.
Pigment Cell Melanoma Res. 2008; 21(5):545-58 [PubMed
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The inactivation of tumor-related genes through the aberrant methylation of promoter CpG islands is thought to contribute to tumor initiation and progression. We therefore investigated promoter methylation events involved in cutaneous melanoma by screening 30 genes of interest for evidence of promoter hypermethylation, examining 20 melanoma cell lines and 40 freshly procured melanoma samples. Utilizing quantitative methylation-specific PCR, we identified five genes (SOCS1, SOCS2, RAR-beta 2, TNFSF10C, and TNFSF10D) with hypermethylation frequencies ranging from 50% to 80% in melanoma cell lines as well as freshly procured tissue samples. Eighteen genes (LOX, RASSF1A, WFDC1, TM, APC, TFPI2, TNFSF10A, CDKN2A, MGMT, TIMP3, ASC, TPM1, IRF8, CIITA-PIV, CDH1, SYK, HOXB13, and DAPK1) were methylated at lower frequencies (2-30%). Two genes (CDKN1B and PTEN), previously reported as methylated in melanoma, and five other genes (RECK, IRF7, PAWR, TNFSF10B, and Rb) were not methylated in the samples screened here. Daughter melanoma cell lines showed identical methylation patterns when compared with original samples from which they were derived, as did synchronous metastatic lesions from the same patient. We identified four genes (TNFSF10C, TNFSF10D, LOX, and TPM1) that have never before been identified as hypermethylated in melanoma, with an overall methylation frequency of 60, 80, 50, and 10%, respectively, hypothesizing that these genes may play an important role in melanoma progression.
Cellular immortalization is one of the prerequisite steps in carcinogenesis. By gene expression profiling, we have found that genes in the interferon (IFN) pathway were dysregulated during the spontaneous cellular immortalization of fibroblasts from Li-Fraumeni syndrome (LFS) patients with germ-line mutations in p53. IFN signaling pathway genes were down-regulated by epigenetic silencing during immortalization, and some of these same IFN-regulated genes were activated during replicative senescence. Bisulfite sequencing of the promoter regions of two IFN regulatory transcription factors (IRF5 and IRF7) revealed that IRF7, but not IRF5, was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The induction of IRF7 gene by IFNalpha in immortal LFS cells was potentiated by pretreatment with the demethylation agent 5-aza-2'-deoxycytidine. Overexpression of IRF5 and IRF7 revealed that they can act either alone or in tandem to activate other IFN-regulated genes. In addition, they serve to inhibit the proliferation rate and induce a senescence-related phenotype in immortal LFS cells. Furthermore, polyinosinic:polycytidylic acid treatment of the IRF-overexpressing cells showed a more rapid induction of several IFN-regulated genes. We conclude that the epigenetic inactivation of the IFN pathway plays a critical role in cellular immortalization, and the reactivation of IFN-regulated genes by transcription factors IRF5 and/or IRF7 is sufficient to induce cellular senescence. The IFN pathway may provide valuable molecular targets for therapeutic interventions at early stages of cancer development.
Chen J, Guo L, Peiffer DA, et al.Genomic profiling of 766 cancer-related genes in archived esophageal normal and carcinoma tissues.
Int J Cancer. 2008; 122(10):2249-54 [PubMed
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We employed the BeadArraytrade mark technology to perform a genetic analysis in 33 formalin-fixed, paraffin-embedded (FFPE) human esophageal carcinomas, mostly squamous-cell-carcinoma (ESCC), and their adjacent normal tissues. A total of 1,432 single nucleotide polymorphisms (SNPs) derived from 766 cancer-related genes were genotyped with partially degraded genomic DNAs isolated from these samples. This directly targeted genomic profiling identified not only previously reported somatic gene amplifications (e.g., CCND1) and deletions (e.g., CDKN2A and CDKN2B) but also novel genomic aberrations. Among these novel targets, the most frequently deleted genomic regions were chromosome 3p (including tumor suppressor genes FANCD2 and CTNNB1) and chromosome 5 (including tumor suppressor gene APC). The most frequently amplified genomic region was chromosome 3q (containing DVL3, MLF1, ABCC5, BCL6, AGTR1 and known oncogenes TNK2, TNFSF10, FGF12). The chromosome 3p deletion and 3q amplification occurred coincidently in nearly all of the affected cases, suggesting a molecular mechanism for the generation of somatic chromosomal aberrations. We also detected significant differences in germline allele frequency between the esophageal cohort of our study and normal control samples from the International HapMap Project for 10 genes (CSF1, KIAA1804, IL2, PMS2, IRF7, FLT3, NTRK2, MAP3K9, ERBB2 and PRKAR1A), suggesting that they might play roles in esophageal cancer susceptibility and/or development. Taken together, our results demonstrated the utility of the BeadArray technology for high-throughput genetic analysis in FFPE tumor tissues and provided a detailed genetic profiling of cancer-related genes in human esophageal cancer.
Craig FE, Johnson LR, Harvey SA, et al.Gene expression profiling of Epstein-Barr virus-positive and -negative monomorphic B-cell posttransplant lymphoproliferative disorders.
Diagn Mol Pathol. 2007; 16(3):158-68 [PubMed
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Although most posttransplant lymphoproliferative disorders (PTLD) are related to Epstein-Barr virus (EBV) infection, approximately 20% lack detectable EBV (EBV-). It is uncertain whether the latter cases are truly distinct from EBV+ PTLD or possibly relate to another infectious agent. This study used gene expression profiling to further investigate the relationship between EBV+ and EBV- monomorphic B-cell PTLD, and to search for clues to their pathogenesis. Affymetrix HU133A GeneChips were used to compare 4 EBV+ and 4 EBV- cases of monomorphic B-cell PTLD. Hierarchical clustering successfully distinguished the EBV+ and EBV- groups. Relative to EBV- PTLD, 54 transcripts were over-expressed in EBV+ PTLD. The transcripts identified included IRF7 (a known regulator of EBV LMP1 expression), EBI2 (EBV-induced gene 2), and 3 that are interferon induced (MX1, IFITM1, and IFITM3). In addition, the EBV+ group contained 232 transcripts decreased relative to the EBV- group, including changes concordant with those previously reported after EBV infection of cultured B-cell lines. In summary, in a small group of monomorphic B-cell PTLD, EBV+ cases demonstrated a subset of gene expression changes associated with EBV infection of B cells. By contrast, EBV- PTLD lacked viral-associated changes suggesting that they are biologically distinct.
Prescott JB, Hall PR, Bondu-Hawkins VS, et al.Early innate immune responses to Sin Nombre hantavirus occur independently of IFN regulatory factor 3, characterized pattern recognition receptors, and viral entry.
J Immunol. 2007; 179(3):1796-802 [PubMed
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Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.
Kaporis HG, Guttman-Yassky E, Lowes MA, et al.Human basal cell carcinoma is associated with Foxp3+ T cells in a Th2 dominant microenvironment.
J Invest Dermatol. 2007; 127(10):2391-8 [PubMed
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Basal cell carcinoma (BCC), the most common human cancer, undergoes spontaneous regression in certain circumstances, which is potentially immune-mediated. To understand the immune response surrounding BCCs, we characterized the genomic, protein, and cellular microenvironment associated with BCC in comparison to normal skin. Our results demonstrated the following: (1) CD4+ CD25+ Foxp3+ surround epithelial tumor aggregates; (2) Immature dendritic cells (DCs) were abundant in the tumor microenvironment; (3) BCC showed increased expression of IL-4, IL-10, and CCL22 and increased expression of interferon-associated genes (IFI27, IRF1, IRF7, and G1P2) and IL-12/23, gene indicating a Th2 dominant microenvironment. Our findings suggest a dynamic state within the immune microenvironment associated with BCC. The finding of phenotypic T regs, in conjunction with immature DCs and Th2 cytokines, suggests an attenuated state of immunity to human BCC. In contrast, abundant CD8+ T cells, an interferon signal, and IL-12/23 suggest partial host antitumor response. A better understanding of these opposing forces within the immune microenvironment may facilitate development of more potent immune-based treatment for BCC and other human carcinomas.
Fukasawa M, Kimura M, Morita S, et al.Microarray analysis of promoter methylation in lung cancers.
J Hum Genet. 2006; 51(4):368-74 [PubMed
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Aberrant DNA methylation is an important event in carcinogenesis. Of the various regions of a gene that can be methylated in cancers, the promoter is the most important for the regulation of gene expression. Here, we describe a microarray analysis of DNA methylation in the promoter regions of genes using a newly developed promoter-associated methylated DNA amplification DNA chip (PMAD). For each sample, methylated Hpa II-resistant DNA fragments and Msp I-cleaved (unmethylated+methylated) DNA fragments were amplified and labeled with Cy3 and Cy5 respectively, then hybridized to a microarray containing the promoters of 288 cancer-related genes. Signals from Hpa II-resistant (methylated) DNA (Cy3) were normalized to signals from Msp I-cleaved (unmethylated+methylated) DNA fragments (Cy5). Normalized signals from lung cancer cell lines were compared to signals from normal lung cells. About 10.9% of the cancer-related genes were hypermethylated in lung cancer cell lines. Notably, HIC1, IRF7, ASC, RIPK3, RASSF1A, FABP3, PRKCDBP, and PAX3 genes were hypermethylated in most lung cancer cell lines examined. The expression profiles of these genes correlated to the methylation profiles of the genes, indicating that the microarray analysis of DNA methylation in the promoter region of the genes is convenient for epigenetic study. Further analysis of primary tumors indicated that the frequency of hypermethylation was high for ASC (82%) and PAX3 (86%) in all tumor types, and high for RIPK3 in small cell carcinoma (57%). This demonstrates that our PMAD method is effective at finding epigenetic changes during cancer.
We have reported evidence for a positive regulatory circuit between interferon regulatory factor 7 (IRF7) and the Epstein-Barr virus (EBV) oncoprotein 1 (LMP1) (S. Ning, A. M. Hahn, and J. S. Pagano, J. Virol. 77:9359-9368, 2003). To explore a possible braking mechanism for this circuit, several type II EBV-infected cell lines that express different levels of LMP1 and IRF7 proteins and therefore are convenient for studying modulation of expression of LMP1 were analyzed. Endogenous levels of IRF7 and LMP1 were directly correlated. Transient expression of an IRF7 dominant-negative mutant decreased LMP1 levels. Endogenous IRF5 and IRF7 proteins were shown to physically associate in EBV-positive cells. Transient expression of IRF5 decreased activation of the LMP1 promoter by IRF7 in a dose-dependent manner. Finally, transfection of either an IRF5 dominant-negative construct or IRF5 small interfering RNA in these cells resulted in increases in endogenous levels of LMP1. These results indicate that IRF5 can downregulate IRF7's induction of expression of LMP1 most likely by interacting with IRF7 and provide a means of modulating a regulatory circuit between IRF7 and LMP1.