Interferons (IFNs) are key players in the tumor immune response and act by inducing the expression of IFN-stimulated genes (ISGs). Here, we identify the mixed-lineage kinase domain-like pseudokinase (MLKL) as an ISG in various cancer cell lines. Both type I and type II IFNs increase the expression of MLKL indicating that MLKL up-regulation is a general feature of IFN signaling. IFNγ up-regulates mRNA as well as protein levels of MLKL demonstrating that IFNγ transcriptionally regulates MLKL. This notion is further supported by Actinomycin D chase experiments showing that IFNγ-stimulated up-regulation of MLKL is prevented in the presence of the transcriptional inhibitor Actinomycin D. Also, knockdown of the transcription factor IFN-regulatory factor 1 (IRF1) and signal transducer and activator of transcription (STAT) 1 as well as knockout of IRF1 significantly attenuate IFNγ-mediated induction of MLKL mRNA levels. Up-regulation of MLKL by IFNγ provides a valuable tool to sensitize cells towards necroptotic cell death and to overcome apoptosis resistance of cancer cells.

OBJECTIVES: We aimed to elucidate the role and molecular mechanisms of FOXM1 in regulating metastasis in oesophageal squamous cell carcinoma (ESCC) as well as its clinical implications.
MATERIALS AND METHODS: The expression levels of four isoforms of FOXM1 were analysed by real-time PCR. Next, genetically modification using overexpression and RNAi systems and transwell were employed to examine FOXM1c function in invasion and migration. Dual luciferase and ChIP assays were performed to decipher the underlying mechanism for transcriptional regulation. The expression levels of FOXM1 and IRF1 were determined by immunohistochemistry staining in ESCC specimens.
RESULTS: The FOXM1c was predominantly overexpressed in ESCC cell lines compared to the other FOXM1 isoforms. Ectopic expression of FOXM1c promoted invasion and migration of ESCC cells lines, whereas downregulation of FOXM1c inhibited these processes. Moreover, FOXM1c expression was positively correlated with IRF1 expression in ESCC cell lines and tumour specimens. IRF1 is, at least in part, responsible for FOXM1c-mediated invasion and migration. Mechanistically, we identified IRF1 as a transcriptional target of FOXM1c and found a FOXM1c-binding site in the IRF1 promoter region. Furthermore, high expression levels of both FOXM1c and IRF1 were positively associated with low survival rate and predicted a poor prognosis of oesophageal cancer patients.
CONCLUSION: FOXM1c promotes the metastasis by transcriptionally targeting IRF1 and may serve as a potential prognostic predictor for oesophageal cancer.

BACKGROUND: Activation of the oncogene YAP has been shown to be related to lung cancer progression and associates with poor prognosis and metastasis. Metformin is a drug commonly used in the treatment of diabetes and with anticancer activity. However, the mechanism through which metformin inhibits tumorigenesis via YAP is poorly understood.
METHODS: The mRNA and protein expressions were analyzed by RT-PCR and western blot. The cellular proliferation was detected by CCK8 and MTT. The cell migration and invasion growth were analyzed by wound healing assay and transwell assay. The activities of promoter were analyzed by luciferase reporter assay. Chromatin immunoprecipitation detected the combining ability of IRF-1 and 5'UTR-YAP.
FINDINGS: Our immunohistochemistry staining and RT-PCR assays showed that the expression of YAP was higher in lung carcinoma samples. Interestingly, metformin was able to downregulate YAP mRNA and protein expression in lung cancer cells. Mechanistically, we found that metformin depressed YAP promoter by competing with the binding of the transcription factor IRF-1 in lung cancer cells. Moreover, combination of metformin and verteporfin synergistically inhibits cell proliferation, promotes apoptosis and suppresses cell migration/invasion by downregulating YAP, therefore reduces the side effects caused by their single use and improve the quality of life for patients with lung cancer.
INTERPRETATION: we concluded that metformin depresses YAP promoter by interfering with the binding of the transcription factor IRF-1. Importantly, verteporfin sensitizes metformin-induced the depression of YAP and inhibition of cell growth and invasion in lung cancer cells. FUND: This work was supported by National Natural Science Foundation of China (No.31801085), the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067 and ZR2017MH125), and Research Foundation of Binzhou Medical University (BY2015KYQD29 and BY2015KJ14).

Transcription factors are recognized as the key regulators of gene expression. However, the changes in the correlation of transcription factors and their target genes between normal and tumor tissues are usually ignored. In this research, we used mRNA expression profile data from The Cancer Genome Atlas which included 5726 samples across 11 major human cancers to perform co-expression analysis by the Pearson correlation coefficients. Then, integrating 81,357 pairs of transcription factors and target genes from transcription factors databases to find out the changes in the co-expression correlation of these gene pairs from normal to tumor tissues. Based on the changes in the number of co-expressed TF-TG pairs and changes in the level of co-expression, we found the generally reduced correlation between transcription factors and their target genes in cancer. Additionally, we screened out universal and specific transcription factors-target genes pairs which may significant influence particular cancer. Then, we obtained 423 cancer cell line expression profiles from Broad Institute Cancer Cell Line Encyclopedia to verify our results. Some of these pairs like XRCC5-XRCC6 have been reported to involve in multiple cancers, while pairs like IRF1-PSMB9 without any previous articles related to tumor but involve in the biological processes of cancer, which are of great potential to be therapeutic targets. Our research may provide insights to better understand the tumor development mechanisms and find potential therapeutic targets.

The adhesion protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is widely expressed in epithelial cells as a short cytoplasmic isoform (S-iso) and in leukocytes as a long cytoplasmic isoform (L-iso) and is frequently silenced in cancer by unknown mechanisms. Previously, we reported that interferon response factor 1 (IRF1) biases alternative splicing (AS) to include the variable exon 7 (E7) in CEACAM1, generating long cytoplasmic isoforms. We now show that IRF1 and a variant of heterogeneous nuclear ribonucleoprotein L (Lv1) coordinately silence the

Interferon regulatory factors (IRFs) are a group of closely related proteins collectively referred to as the IRF family. Members of this family were originally recognized for their roles in inflammatory responses; however, recent research has suggested that they are also involved in tumor biology. This review focusses on current knowledge of the roles of IRF-1 and IRF-2 in human cancer, with particular attention paid to the impact of IRF-1 inactivation. The different mechanisms underlying IRF-1 inactivation and their implications for human cancers and the potential importance of IRF-1 in immunotherapy are also summarized.

Programmed death-ligand 1 (PD-L1) acts as an immune checkpoint inhibitor in various cancers. PD-L1 is known to be more frequently expressed in EBV (+) gastric cancer (GC). However, the mechanisms underlying the regulation of PD-L1 expression in EBV (+) GC remain unclear. We investigated the basal and inducible PD-L1 expressions in GC cells. PD-L1 expression was upregulated upon treatment with IFNγ in both EBV (-) and EBV (+) GC cells. Upon stimulation with the same concentration of IFNγ for 24 h, EBV (+) SNU-719 cells showed dramatically higher PD-L1 expression levels by activating JAK2/STAT1/IRF-1 signaling than those of EBV (-) AGS cells. PD-L1 promoter assays, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that IFNγ-inducible PD-L1 overexpression is primarily mediated by the putative IRF-1α site of the PD-L1 promoter in EBV (+) SNU-719 cells. Moreover, EBNA1 knockdown reduced both constitutive and IFNγ-inducible PD-L1 promoter activity by decreasing the transcript and protein levels of JAK2 and subsequently STAT1/IRF-1/PD-L1 signaling. EBNA1 is suggested to be moderately enhance both constitutive and IFNγ-inducible PD-L1 expression in EBV (+) GC cells. Thus, the signaling proteins and EBNA1 that regulate PD-L1 expression are potential therapeutic targets in EBV (+) GC.

Retinoids, derivatives of vitamin A, are key physiological molecules with regulatory effects on cell differentiation, proliferation and apoptosis. As a result, they are of interest for cancer therapy. Specifically, models of breast cancer have varied responses to manipulations of retinoid signaling. This study characterizes the transcriptional response of MDA-MB-231 and MDA-MB-468 breast cancer cells to retinaldehyde dehydrogenase 1A3 (ALDH1A3) and all-trans retinoic acid (atRA). We demonstrate limited overlap between ALDH1A3-induced gene expression and atRA-induced gene expression in both cell lines, suggesting that the function of ALDH1A3 in breast cancer progression extends beyond its role as a retinaldehyde dehydrogenase. Our data reveals divergent transcriptional responses to atRA, which are largely independent of genomic retinoic acid response elements (RAREs) and consistent with the opposing responses of MDA-MB-231 and MDA-MB-468 to in vivo atRA treatment. We identify transcription factors associated with each gene set. Manipulation of the IRF1 transcription factor demonstrates that it is the level of atRA-inducible and epigenetically regulated transcription factors that determine expression of target genes (e.g. CTSS, cathepsin S). This study provides a paradigm for complex responses of breast cancer models to atRA treatment, and illustrates the need to characterize RARE-independent responses to atRA in a variety of models.

BACKGROUND: Recently, neoadjuvant chemotherapy with docetaxel/cisplatin/5-fluorouracil (NAC-DCF) was identified as a novel strong regimen with a high rate of pathological complete response (pCR) in advanced esophageal cancer in Japan. Predicting pCR will contribute to the therapeutic strategy and the prevention of surgical invasion. However, a predictor of pCR after NAC-DCF has not yet been developed. The aim of this study was to identify a novel predictor of pCR in locally advanced esophageal cancer treated with NAC-DCF.
PATIENTS AND METHODS: A total of 32 patients who received NAC-DCF followed by esophagectomy between June 2013 and March 2016 were enrolled in this study. We divided the patients into the following 2 groups: pCR group (9 cases) and non-pCR group (23 cases), and compared gene expressions between these groups using DNA microarray data and KeyMolnet. Subsequently, a validation study of candidate molecular expression was performed in 7 additional cases.
RESULTS: Seventeen molecules, including transcription factor E2F, T-cell-specific transcription factor, Src (known as "proto-oncogene tyrosine-protein kinase of sarcoma"), interferon regulatory factor 1, thymidylate synthase, cyclin B, cyclin-dependent kinase (CDK) 4, CDK, caspase-1, vitamin D receptor, histone deacetylase, MAPK/ERK kinase, bcl-2-associated X protein, runt-related transcription factor 1, PR domain zinc finger protein 1, platelet-derived growth factor receptor, and interleukin 1, were identified as candidate molecules. The molecules were mainly associated with pathways, such as transcriptional regulation by SMAD, RB/E2F, and STAT. The validation study indicated that 12 of the 17 molecules (71%) matched the trends of molecular expression.
CONCLUSIONS: A 17-molecule set that predicts pCR after NAC-DCF for locally advanced esophageal cancer was identified.

Interferon regulatory factor 1 (IRF1) has been suggested to act as a tumor suppressor in human cancers. However, the clinical significance and biological function of IRF1 in cholangiocarcinoma is poorly understood. In our results, IRF1 mRNA and protein expressions were decreased in cholangiocarcinoma tissues and cell lines compared with paired normal hepatic tissues and intrahepatic bile duct epithelial cell line. IRF1 protein low-expression was associated with tumor stage, tumor size, vascular invasion and metastasis and served as a poor independent prognostic parameter in cholangiocarcinoma patients. Up-regulation of IRF1 expression suppressed cholangiocarcinoma cells proliferation, migration and invasion, and blocked cell cycle progression, but has no effect on apoptosis. In conclusion, IRF1 is low-expressed in cholangiocarcinoma tissues and cell lines, and correlated with malignant status and prognosis in cholangiocarcinoma patients. IRF1 served as tumor suppressor in the regulation of cholangiocarcinoma cells proliferation, cell cycle, migration and invasion.

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1

Since cancer cells often evade apoptosis, induction of necroptosis as another mode of programmed cell death is considered a promising therapeutic alternative. Here, we identify a novel synergistic interaction of Smac mimetics that antagonize x-linked Inhibitor of Apoptosis (XIAP), cellular Inhibitor of Apoptosis (cIAP) 1 and 2 with interferon (IFN)γ to induce necroptosis in apoptosis-resistant cancer cells in which caspase activation is blocked. This synergism is confirmed by calculation of combination indices (CIs) and found in both solid and hematological cancer cell lines as well as for different Smac mimetics (i.e. BV6, Birinapant), pointing to a broader relevance. Importantly, individual genetic knockdown of key components of necroptosis signaling, i.e. receptor-interacting protein (RIP) 1, RIP3 or mixed lineage kinase domain-like pseudokinase (MLKL), significantly protects from BV6/IFNγ-induced cell death. Similarly, pharmacological inhibitors of RIP1 (necrostatin-1(Nec-1)), RIP3 (GSK'872) or MLKL (necrosulfonamide (NSA)) significantly reduce BV6/IFNγ-stimulated cell death. Of note, IFN-regulatory factor (IRF)1 is required for BV6/IFNγ-mediated necroptosis, as IRF1 silencing provides protection from cell death. By comparison, antibodies blocking tumor necrosis factor (TNF)α, TNF-related apoptosis-inducing ligand (TRAIL) or CD95 ligand fail to inhibit BV6/IFNγ-induced cell death, pointing to a mechanism independently of death receptor ligands. This is the first report showing that Smac mimetics synergize with IFNγ to trigger necroptosis in apoptosis-resistant cancer cells with important implications for Smac mimetic-based strategies for the treatment of cancer.

BACKGROUND: Neuroendocrine-differentiated prostate cancer (NEPCa) is refractory to androgen deprivation therapy and shows a poor prognosis. The underlying mechanisms responsible for neuroendocrine differentiation (NED) are yet to be clarified. In this study, we investigated the role of mammalian target of rapamycin (mTOR) in NEPCa.
METHODS: We utilized a gain-of-function analysis by establishing a human PCa LNCaP stable line that expresses hyperactive mTOR (LNCaP-mTOR). Then, we employed a comprehensive mass spectrometric analysis to identify a key transcription factor in LNCaP-mTOR, followed by a loss-of-function analysis using CRISPR/Cas system.
RESULTS: The activation of mTOR induced NED. We observed significant cell growth arrest in NED of LNCaP-mTOR, which accompanied increased expression of p21
CONCLUSIONS: We identified active mTOR as a novel inducer of NED, and elucidated a mechanism underlying the malignant transformation of NEPCa by recapitulating NED in vitro.

The synergistic activation mediator (SAM) system can robustly activate endogenous gene expression by a single-guide RNA. This transcriptional modulation has been shown to enhance gene promoter activity and leads to epigenetic changes. Human interferon-γ is a common natural glycoprotein involved in antiviral effects and inhibition of cancer cell growth. Large quantities of high-purity interferon-γ are important for medical research and clinical therapy. To investigate the possibility of employing the SAM system to enhance endogenous human interferon-γ with normal function in innate immunity, we designed 10 single-guide RNAs that target 200 bp upstream of the transcription start sites of the interferon-γ genome, which could significantly activate the interferon-γ promoter reporter. We confirmed that the system can effectively and highly activate interferon-γ expression in several humanized cell lines. Moreover, we found that the interferon-γ induced by the SAM system could inhibit tumorigenesis. Taken together, our results reveal that the SAM system can modulate epigenetic traits of non-immune cells through activating interferon-γ expression and triggering JAK-STAT signaling pathways. Thus, this strategy could offer a novel approach to inhibit tumorigenesis without using exogenous interferon-γ.

PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression.

BACKGROUND: Predictive biomarkers for antibodies against programmed death 1 (PD-1) remain a major unmet need in metastatic melanoma. Specifically, response is seen in tumors that do not express programmed death ligand 1 (PD-L1), highlighting the need for a more sensitive biomarker. We hypothesize that capacity to express PD-L1, as assessed by an assay for a PD-L1 transcription factor, interferon regulatory factor 1 (IRF-1), may better distinguish patients likely to benefit from anti-PD-1 immunotherapy.
METHODS: Samples from 47 melanoma patients that received nivolumab, pembrolizumab, or combination ipilimumab/nivolumab at Yale New Haven Hospital from May 2013 to March 2016 were collected. Expression of IRF-1 and PD-L1 in archival pre-treatment formalin-fixed, paraffin-embedded tumor samples were assessed by the AQUA method of quantitative immunofluorescence. Objective radiographic response (ORR) and progression-free survival (PFS) were assessed using modified RECIST v1.1 criteria.
RESULTS: Nuclear IRF-1 expression was higher in patients with partial or complete response (PR/CR) than in patients with stable or progressive disease (SD/PD) (
CONCLUSIONS: As a measure of PD-L1 expression capability, IRF-1 expression may be a more valuable predictive biomarker for anti-PD-1 therapy than PD-L1 itself.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

Interferon-α (IFN-α) and IFN-λ are structurally distinct cytokines that bind to different receptors, but induce expression of similar sets of genes through Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. The difference between IFN-α and IFN-λ signaling remains poorly understood. Here, using the CRISPR/Cas9 system, we examine the role of STAT1 and STAT2 in the inhibition of hepatitis C virus (HCV) replication by IFN-α and IFN-λ. Treatment with IFN-α increases expression of IFN-stimulated genes (ISGs) such as double-stranded RNA-activated protein kinase (PKR) and decreases viral RNA and protein levels in HCV-infected Huh-7.5 human hepatoma cells. These responses are only partially attenuated by knockout of STAT1 but are abolished by knockout of STAT2. In contrast, the inhibition of HCV replication by IFN-λ is abolished by knockout of STAT1 or STAT2. Microarray analysis reveals that IFN-α but not IFN-λ can induce expression of the majority of ISGs in STAT1 knockout cells. These findings suggest that IFN-α can inhibit HCV replication through a STAT2-dependent but STAT1-independent pathway, whereas IFN-λ induces ISG expression and inhibits HCV replication exclusively through a STAT1- and STAT2-dependent pathway.

BACKGROUND/AIMS: Interferon regulatory factor 1 (IRF-1) has been shown to function as a transcriptional activator or repressor of a variety of target genes. However, its upstream, non-coding RNA-related regulatory capacity remains unknown. In this study, we focus on the miRNA-associated single nucleotide polymorphisms (SNPs) in the 3'untranslated region (UTR) of IRF-1 to further investigate the functional relationship and potential diagnostic value of the SNPs and miRNAs among Chinese gastric cancer (GC) patients.
METHODS: We performed a case-control study with 819 GC patients and 756 cancer-free controls. Genotyping by realtime PCR assay, cell transfection, and the dual luciferase reporter assay were used in our study, and the 5-year overall survival rate and relapse-free survival rate in different groups were investigated.
RESULTS: We found that patients suffering from Helicobacter pylori (Hp) infection were the susceptible population compared to controls. SNP rs56288038 (C/G) in IRF-1 3'UTR was involved in the occurrence of GC by acting as a tumor promoter factor. SNP rs56288038 (C/G) could be up-regulated by miR-502-5p, which caused a down-regulation of IRF-1 in cell lines and decreased apoptosis induced by IFN-γ. Carrying the G genotype was related to significantly low expression of IRF-1 and Hp infection, poor differentiation, big tumor size, invasion depth, as well as the high probability of metastasis, and moreover, the C/G SNP was associated with shorter survival of GC patients with five years of follow-up study.
CONCLUSIONS: our findings have shown that the SNP rs56288038 (C/G) in IRF-1 3'UTR acted as a promotion factor in GC development through enhancing the regulatory role of miR-502-5p in IRF-1 expression.

The Wilms tumor suppressor, WT1 was first identified due to its essential role in the normal development of the human genitourinary system. Wilms tumor 1 associated protein (WTAP) was subsequently revealed to interact with WT1 using yeast two-hybrid screening. The present study identified 44 complete WTAP genes in the genomes of vertebrates, including fish, amphibians, birds and mammals. The vertebrate WTAP proteins clustered into the primate, rodent and teleost lineages using phylogenetic tree analysis. From 1,347 available SNPs in the human WTAP gene, 19 were identified to cause missense mutations. WTAP was expressed in bladder, blood, brain, breast, colorectal, esophagus, eye, head and neck, lung, ovarian, prostate, skin and soft tissue cancers. A total of 17 out of 328 microarrays demonstrated an association between WTAP gene expression and cancer prognosis. However, the association between WTAP gene expression and prognosis varied in distinct types of cancer, and even in identical types of cancer from separate microarray databases. By searching the Catalogue of Somatic Mutations in Cancer database, 65 somatic mutations were identified in the human WTAP gene from the cancer tissue samples. These results suggest that the function of WTAP in tumor formation may be multidimensional. Furthermore, signal transducer and activator of transcription 1, forkhead box protein O1, interferon regulatory factor 1, glucocorticoid receptor and peroxisome proliferator-activated receptor γ transcription factor binding sites were identified in the upstream (promoter) region of the human WTAP gene, suggesting that these transcription factors may be involved in WTAP functions in tumor formation.

BACKGROUND: The long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) plays a crucial role in cancer progression, which is regulated by the interferon regulatory factor-1 (IRF1) and up-streaming Akt activation. The present study evaluated the chemopreventive effects of delphinidin-3-glucoside (Dp), a major anthocyanin present in pigmented fruits and vegetables, on breast carcinogenesis, and investigate the role of the Akt/HOTAIR signaling pathway.
METHODS: Human breast epithelial cells MCF10A were treated with carcinogens (NNK and B[a]P) or co-treated with carcinogens plus Dp for 30 days. Then, the cancer-associated properties of the treated cells were evaluated to assess the carcinogenesis and the effects of Dp. HOTAIR levels were detected by qRT-PCR. The proteins expression was measured by western blots, immunofluorescence and immunohistochemistry. Xenografted tumors were made by implanting breast cancer cells MDA-MB-231-Luc-GFP in athymic mice. ChIP-qPCR analysis was used to detect the IRF1 binding to the HOTAIR promoter.
RESULTS: Carcinogens treatment induces apparent carcinogenic transformation in MCF10A cells including reduced dependence on growth factors, anchorage-independent cell growth and aberrant wound-healing ability, which is effectively suppressed by Dp co-treatment. The level of HOTAIR significantly increases in a time-dependent manner during chronic breast carcinogenesis. Dp treatment down-regulates HOTAIR expression in breast carcinogenesis and breast cancer cells. Furthermore, Dp administration inhibits the growth of xenografted breast tumors in athymic mice, and decreases HOTAIR in vivo. Further studies showed that Dp represses Akt activation, promotes IRF1 expression and increases IRF1 binding to the HOTAIR promoter. Silence of IRF1 expression via transfecting cells with IRF1 siRNAs significantly reduced the effects of Dp on HOTAIR, resulting in decreased cytotoxic effects of Dp on breast cancer cells.
CONCLUSIONS: These data suggest the effective chemopreventive effect of Dp on breast carcinogenesis, in which down-regulation of HOTAIR plays a critical role.

Objective To investigate the effect of endogenous interferon β (IFN-β) on the polarization of M1 macrophages as well as the proliferation and invasion activities of hepatocellular carcinoma cells (HCCs) mediated by M1 macrophages. Methods U937-M1 macrophages derived from human monocytic tumor cells U937 was established and the cell phenotypes were identified by real-time quantitative PCR, ELISA and flow cytometry. After IFN-β gene was knocked down with siRNA or IFN-β was neutralized with IFN-β monoantibody in U937-M1 macrophages, the change of M1/M2 phenotype was again analyzed by the above methods. The expressions of interferon regulatory factor 1 (IRF1) and IRF5 were detected by real-time quantitative PCR and Western blotting. The proliferation and invasion activities of HCCs, which were cultured with conditioned medium (CM) collected from different macrophage groups, were analyzed by CCK-8 assay and Transwell(TM) experiments, respectively. Results U937-M1 macrophages showed higher expressions of interleukin 12p35 (IL-12p35), interleukin 12p40 (IL-12p40), interleukin 12p70 (IL-12p70), interleukin 23p19 (IL-23p19), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and CD86 than U937-M0 did. But both U937-M0 macrophages and U937-M1 macrophages showed low expression of CD206. However, compared with the U937-M1 macrophages, the IFN-β-blocked U937-M1 macrophages presented decreased expressions of the above M1 macrophages-associated markers, but increased expressions of M2 macrophages-associated markers IL-10 and CD206, as well as lower expressions of IRF1 and IRF5. The inhibited proliferation/invasion activities of HCCs mediated by U937-M1 macrophages were reversed by IFN-β-blocked U937-M1 macrophages. Conclusion Blocking endogenous IFN-β could inhibit the U937-M1 polarization status and U937-M1 macrophages-mediated anti-tumor activity of HCCs. IFN-β might be involved in modulating the expressions of IRF1 and IRF5 as well as maintaining the M1 polarization status and its function.

Interferon regulatory factor-1 (IRF-1) is a tumor-suppressor gene induced by interferon-γ (IFNγ) and plays an important role in the cell death of hepatocellular carcinoma (HCC). HCC tumors evade death in part by downregulating IRF-1 expression, yet the molecular mechanisms accounting for IRF-1 suppression in HCC have not yet been characterized. Previous studies have shown that microRNA-23a (miR-23a) can suppress apoptosis by targeting IRF-1. Therefore, we hypothesized that miR-23a promotes HCC growth by downregulating IRF-1. For the in vivo studies, 7 cases of resected HCC and adjacent liver samples were analyzed. For the in vitro studies, IRF-1 mRNA and protein were examined in HepG2 and Huh-7 HCC cells after IFNγ stimulation by real-time PCR and western blotting, respectively. To determine the role of miR-23a in regulating IRF-1, HepG2 cells were transfected with an miR-23a mimic or inhibitor, and IRF-1 expression was examined. Binding of miR-23a was assessed by cloning the 528-bp human IRF-1 3'-untranslated region (3'UTR) into luciferase reporter plasmid pMIR-IRF-1-3'UTR. The results showed that IRF-1 mRNA expression was downregulated in the human HCC tumor tissues compared to that in the adjacent background liver tissues. IFNγ-induced IRF-1 protein was less in the HepG2 tumor cells compared to that in the primary human hepatocytes. miR-23a expression was inversely correlated with IRF-1, and addition of the miR-23a inhibitor increased basal IRF-1 mRNA and protein. Likewise, the miR-23a mimic downregulated IFNγ-induced IRF-1 protein expression, while the miR-23a inhibitor increased IRF-1. Furthermore, the miR-23a mimic repressed IRF-1-3'UTR reporter activity, while the miR-23a inhibitor increased the reporter activity. These results demonstrated that IRF-1 expression is downregulated in human HCC tumors compared to that noted in the background liver. miR-23a downregulates the expression of IRF-1 in HCC cells, and the IRF-1 3'UTR has an miR‑23a binding site that binds miR-23a and decreases reporter activity. These findings suggest that the targeting of IRF-1 by miR-23a may be the molecular basis for IRF-1 downregulation in HCC and provide new insight into the regulation of HCC by miRNAs.

Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer.

Both oncogenic and tumor-suppressor activities are attributed to the Nuclear Factor kappa B (NF-kB) pathway. Moreover, NF-kB may positively or negatively regulate proliferation. The molecular determinants of these opposing roles of NF-kB are unclear. Using primary human mammary epithelial cells (HMEC) as a model, we show that increased RelA levels and consequent increase in basal transcriptional activity of RelA induces IRF1, a target gene. Induced IRF1 upregulates STAT1 and IRF7, and in consort, these factors induce the expression of interferon response genes. Activation of the interferon pathway down-regulates CDK4 and up-regulates p27 resulting in Rb hypo-phosphorylation and cell cycle arrest. Stimulation of HMEC with IFN-γ elicits similar phenotypic and molecular changes suggesting that basal activity of RelA and IFN-γ converge on IRF1 to regulate proliferation. The anti-proliferative RelA-IRF1-CDK4 signaling axis is retained in ER+/HER2- breast tumors analyzed by The Cancer Genome Atlas (TCGA). Using immuno-histochemical analysis of breast tumors, we confirm the negative correlation between RelA levels and proliferation rate in ER+/HER2- breast tumors. These findings attribute an anti-proliferative tumor-suppressor role to basal RelA activity. Inactivation of Rb, down-regulation of RelA or IRF1, or upregulation of CDK4 or IRF2 rescues the RelA-IRF1-CDK4 induced proliferation arrest in HMEC and are points of disruption in aggressive tumors. Activity of the RelA-IRF1-CDK4 axis may explain favorable response to CDK4/6 inhibition observed in patients with ER+ Rb competent tumors.

BACKGROUND: Cancers adapt to immune-surveillance through evasion. Immune responses against carcinoma and melanoma converge on cytotoxic effectors and IFNγ-STAT1-IRF1 signalling. Local IFN-driven immune checkpoint expression can mediate feedback inhibition and adaptive immune resistance. Whether such coupled immune polarization and adaptive resistance is generalisable to lymphoid malignancies is incompletely defined. The host response in diffuse large B-cell lymphoma (DLBCL), the commonest aggressive lymphoid malignancy, provides an empirical model.
METHODS: Using ten publicly available gene expression data sets encompassing 2030 cases we explore the nature of host response in DLBCL. Starting from the "cell of origin" paradigm for DLBCL classification, we use the consistency of differential expression to define polarized patterns of immune response genes in DLBCL, and derive a linear classifier of immune response gene expression. We validate and extend the results in an approach independent of "cell of origin" classification based on gene expression correlations across all data sets.
RESULTS: T-cell and cytotoxic gene expression with polarization along the IFNγ-STAT1-IRF1 axis provides a defining feature of the immune response in DLBCL. This response is associated with improved outcome, particularly in the germinal centre B-cell subsets of DLBCL. Analysis of gene correlations across all data sets, independent of "cell of origin" class, demonstrates a consistent association with a hierarchy of immune-regulatory gene expression that places IDO1, LAG3 and FGL2 ahead of PD1-ligands CD274 and PDCD1LG2.
CONCLUSION: Immune responses in DLBCL converge onto the IFNγ-STAT1-IRF1 axis and link to diverse potential mediators of adaptive immune resistance identifying future therapeutic targets.

PURPOSE: Antiangiogenic therapy is commonly being used for the treatment of glioblastoma. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. Determining the mechanism of treatment failure of the VEGF monoclonal antibody bevacizumab for malignant glioma would provide insight into approaches to overcome therapeutic resistance.
EXPERIMENTAL DESIGN: In this study, we evaluated the effects of bevacizumab on the autophagy of glioma cells and determined target genes involving in the regulation of bevacizumab-induced autophagy.
RESULTS: We demonstrated that bevacizumab treatment increased expression of autophagy markers and autophagosome formation in cell culture experiments as well as in in vivo studies. Gene expression profile analysis performed on murine xenograft models of glioblastoma showed increased transcriptional levels of STAT1/IRF1 signaling in bevacizumab resistant tumors compared to control tumors. In vitro experiments showed that bevacizumab treatment increased IRF1 expression in a dose and time dependent manner, which was coincident with bevacizumab-mediated autophagy. Down regulation of IRF1 by shRNA blocked autophagy and increased AIF-dependent apoptosis in bevacizumab-treated glioma cells. Consistently, IRF1 depletion increased the efficacy of anti-VEGF therapy in a glioma xenograft model, which was due to less bevacizumab-promoted autophagy and increased apoptosis in tumors with down-regulated IRF1.
CONCLUSIONS: These data suggest that IRF1 may regulate bevacizumab-induced autophagy, and may be one important mediator of glioblastoma resistant to bevacizumab.

BACKGROUND/AIM: Uterine leiomyosarcoma (Ut-LMS) is a highly metastatic smooth muscle neoplasm. We have previously reported that low molecular mass protein2 Lmp2-deficient mice spontaneously developed Ut-LMS, which implicated this protein as an anti-oncogenic candidate. We also suggested that LMP2 may negatively regulate Ut-LMS independently of its role in the proteasome. Initially described as a transcription factor able to activate the expression of interferon-gamma (IFN-γ)-responsive genes, interferon regulatory factor-1 (IRF1) has been shown to play roles in the immune response, and tumor suppression. The aim of this study was to elucidate the molecular mechanism of sarcomagenesis of Ut-LMS using human and mouse uterine tissues.
MATERIALS AND METHODS: The expression of the IFN-γ signal molecules, IRF1 and -2, STAT1, and LMP2, -3, -7 and -10 were examined by western blot analysis, electrophoretic mobility shift assay and immunohistochemistry in human and mouse uterine tissues. Physiological significance of IRF1 in sarcomagenesis of Ut-LMS was demonstrated by xenograft studies.
RESULTS: In the present study, several lines of evidence indicated that although treatment with IFN-γ strongly induced the activation of STAT1 as a transcriptional activator, its target molecule, IRF1, was not clearly produced in Lmp2-deficient uterine smooth muscle cells (Ut-SMCs).
CONCLUSION: Defective expression of IRF1 in the IFN-γ-induced signaling molecules may result in the malignant transformation of Ut-SMCs. The modulation of LMP2 may lead to new therapeutic approaches in human Ut-LMS.

Interferon Regulatory Factor (IRF)-1, originally identified as a transcription factor of the human interferon (IFN)-β gene, mediates tumor suppression and may inhibit oncogenesis. We have shown that IRF-1 in human breast cancer cells results in the down-regulation of survivin, tumor cell death, and the inhibition of tumor growth in vivo in xenogeneic mouse models. In this current report, we initiate studies comparing the effect of IRF-1 in human nonmalignant breast cell and breast cancer cell lines. While IRF-1 in breast cancer cells results in growth inhibition and cell death, profound growth inhibition and cell death are not observed in nonmalignant human breast cells. We show that TNF-α or IFN-γ induces IRF-1 in breast cancer cells and results in enhanced cell death. Abrogation of IRF-1 diminishes TNF-α and IFN-γ-induced apoptosis. We test the hypothesis that IRF-1 augments TNF-α-induced apoptosis in breast cancer cells. Potential signaling networks elicited by IRF-1 are investigated by evaluating the NF-κB pathway. TNF-α and/or IFN-γ results in decreased presence of NF-κB p65 in the nucleus of breast cancer cells. While TNF-α and/or IFN-γ can induce IRF-1 in nonmalignant breast cells, a marked change in NF-κB p65 is not observed. Moreover, the ectopic expression of IRF-1 in breast cancer cells results in caspase-3, -7, -8 cleavage, inhibits NF-κB activity, and suppresses the expression of molecules involved in the NF-κB pathway. These data show that IRF-1 in human breast cancer cells elicits multiple signaling networks including intrinsic and extrinsic cell death and down-regulates molecules involved in the NF-κB pathway.