Immune surveillance of brain metastatic cancer cells is mediated by IFITM1.

Brain metastasis, most commonly originating from lung cancer, increases cancer morbidity and mortality. Although metastatic colonization is the rate-limiting and most complex step of the metastatic cascade, the underlying mechanisms are poorly understood. Here, in vivo genome-wide CRISPR-Cas9 screening revealed that loss of interferon-induced transmembrane protein 1 (IFITM1) promotes brain colonization of human lung cancer cells. Incipient brain metastatic cancer cells with high expression of IFITM1 secrete microglia-activating complement component 3 and enhance the cytolytic activity of CD8+ T cells by increasing the expression and membrane localization of major histocompatibility complex class I. After activation, microglia (of the innate immune system) and cytotoxic CD8+ T lymphocytes (of the adaptive immune system) were found to jointly eliminate cancer cells by releasing interferon-gamma and inducing phagocytosis and T-cell-mediated killing. In human cancer clinical trials, immune checkpoint blockade therapy response was significantly correlated with IFITM1 expression, and IFITM1 enhanced the brain metastasis suppression efficacy of PD-1 blockade in mice. Our results exemplify a novel mechanism through which metastatic cancer cells overcome the innate and adaptive immune responses to colonize the brain, and suggest that a combination therapy increasing IFITM1 expression in metastatic cells with PD-1 blockade may be a promising strategy to reduce metastasis.

Type 1 Interferon Responses Underlie Tumor-Selective Replication of Oncolytic Measles Virus.

The mechanism of tumor-selective replication of oncolytic measles virus (MV) is poorly understood. Using a stepwise model of cellular transformation, in which oncogenic hits were additively expressed in human bone marrow-derived mesenchymal stromal cells, we show that MV-induced oncolysis increased progressively with transformation. The type 1 interferon (IFN) response to MV infection was significantly reduced and delayed, in accordance with the level of transformation. Consistently, we observed delayed and reduced signal transducer and activator of transcription (STAT1) phosphorylation in the fully transformed cells. Pre-treatment with IFNß restored resistance to MV-mediated oncolysis. Gene expression profiling to identify the genetic correlates of susceptibility to MV oncolysis revealed a dampened basal level of immune-related genes in the fully transformed cells compared to their normal counterparts. IFN-induced transmembrane protein 1 (IFITM1) was the foremost basally downregulated immune gene. Stable IFITM1 overexpression in MV-susceptible cells resulted in a 50% increase in cell viability and a significant reduction in viral replication at 24 h after MV infection. Overall, our data indicate that the basal reduction in functions of the type 1 IFN pathway is a major contributor to the oncolytic selectivity of MV. In particular, we have identified IFITM1 as a restriction factor for oncolytic MV, acting at early stages of infection.

Expression of IFN-induced transmembrane protein 1 in glomerular endothelial cells.

BACKGROUND: Glomerular endothelial cells (GECs) are directly exposed to circulating viral particles in the glomerulus. Although viral infections may trigger the development of acute kidney injury or the worsening of pre-existing chronic kidney disease, the specific molecular mechanisms underlying antiviral reactions via the activation of endothelial Toll-like receptor 3 signaling in the kidney remain to be determined. Interferon (IFN)-induced transmembrane protein 1 (IFITM1), a member of interferon-stimulated gene protein family, is involved in the prevention of viral entry into cerebral vascular endothelial cells, respiratory epithelial cells, and endometrium. However, as far as we are aware, the implication of IFITM1 associated with viral infections in GECs has not been investigated to date. METHODS: Cultured, normal human GECs were treated with polyinosinic-polycytidylic acid (poly IC), a synthesized viral double-stranded RNA, then the expression of IFITM1 was examined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. To further elucidate the poly IC-induced signaling pathway, the cells were applied to RNA interference against IFN-ß, nuclear factor-κB p65, and IFN regulatory factor 3. We also conducted an immunofluorescence study to examine endothelial IFITM1 expression in biopsy specimens from patients with chronic kidney disease. RESULTS: We found that the activation of Toll-like receptor 3 induced endothelial expression of IFITM1, and that this involved IFN regulatory factor 3 and IFN-ß, but not nuclear factor-κB. Intense endothelial IFITM1 immunoreactivity was observed in biopsy specimens from patients with lupus nephritis. CONCLUSIONS: Antiviral reaction-related endothelial expression of IFITM1 may be involved, at least in part, in the development of particularly in lupus nephritis. Further detailed studies of the implication of interferon stimulated genes, including IFITM1 in GECs are needed.

Knockdown of interferon-induced transmembrane protein 1 inhibited proliferation, induced cell cycle arrest and apoptosis, and suppressed MAPK signaling pathway in pancreatic cancer cells.

Pancreatic cancer (PC), highly malignant, is one of the most lethal cancers. Interferon-induced transmembrane protein 1 (IFITM1) has recently been regarded as a new molecular marker in human cancers. However, the role of IFITM1 in PC remains unclear. In this study, a short hairpin RNA (shRNA) was constructed to assess the effect of IFITM1 on PANC-1 and ASPC-1 cells. The level of IFITM1 was downregulated in cells transfected with shRNA targeting IFITM1 (sh-IFITM1). Silencing of IFITM1 significantly decreased cell viability, downregulated the level of Ki-67, arrested cell at G1/S phase, reduced the number of cells in S phase, and decreased cyclinD1, cyclinE, CDK2, and CDK4 levels. Moreover, Hoechst staining and Western blotting analysis showed that cell apoptosis was induced by IFITM1. IFITM1 knockdown suppressed the MAPK signaling pathway by downregulation of p-ERK, p-P38, and p-JNK levels. These findings suggested that IFITM1 could be considered a potential therapeutic target for PC.

γ-Secretase inhibition affects viability, apoptosis, and the stem cell phenotype of endometriotic cells.

INTRODUCTION: Stem cells mediate cyclic regeneration of the endometrium. The upregulated expression of receptors and modulators of the notch signaling pathway in endometriosis suggests an involvement in the pathogenetic process. Here, we investigated the effects of notch pathway inhibition by a γ-secretase inhibitor (GSI) on stemness-associated properties of the epithelial endometriotic cell line 12Z and of primary endometriotic stroma cells. MATERIAL AND METHODS: 12Z cells and primary endometriotic stroma cells of 7 patients were treated with or without GSI, and analyzed for changes in gene expression by TaqMan low-density arrays, quantitative PCR, and flow cytometry. The functional impact of GSI treatment was studied by MTT assay, cell cycle analysis, colony formation assay, annexin V apoptosis assay, and aldehyde dehydrogenase activity assays. RESULTS: In 12Z cells, GSI treatment reduced aldehyde dehydrogenase activity and colony formation, and induced a shift to the G2/M phase of the cell cycle. Cell viability was decreased and apoptosis was increased in both cell models. GSI further induced transcriptional downregulation of the stemness-associated factors leukemia inhibitory factor receptor (LIFR), sex-determining region Y (SRY)- box 2, interferon-induced transmembrane protein 1, and hes-related family bHLH transcription factor with YRPW motif 1, in 12Z cells and in primary cell cultures. Downregulation of LIFR expression by GSI was confirmed at the protein level by flow cytometry. CONCLUSIONS: Our in vitro data suggest that application of GSI may be a worthwhile approach in the treatment of endometriosis that warrants further investigation.

Oncostatin M Induces IFITM1 Expression to Inhibit Hepatitis B Virus Replication Via JAK-STAT Signaling.

BACKGROUND & AIMS: Functional cure is achieved by a limited number of patients with chronic hepatitis B (CHB) after nucleotide analogue(s) and interferon treatment. It is urgent to develop therapies that can help a larger proportion of patients achieve functional cure. The present study was designed to explore the anti-hepatitis B virus (HBV) potency of interleukin-6 family cytokines and to characterize the underlying mechanisms of the cytokine displaying the highest anti-HBV potency. METHODS: HBV-infected cells were used to screened the anti-HBV potency of interleukin-6 family cytokines. The concentration of oncostatin M (OSM) in patients with chronic HBV infection was examined by enzyme-linked immunosorbent assay. The underlying mechanism of OSM anti-HBV was explored through RNA-seq. C57BL/6 mice injected with rAAV8-1.3HBV were used to explore the suppression effect of OSM on HBV in vivo. RESULTS: OSM is the most effective of the interleukin-6 family cytokines for suppression of HBV replication (percentage of average inhibition: hepatitis B surface antigen, 34.44%; hepatitis B e antigen, 32.52%; HBV DNA, 61.57%). Hepatitis B e antigen-positive CHB patients with high OSM levels had lower hepatitis B surface antigen and hepatitis B e antigen than those with low levels. OSM activated JAK-STAT signaling pathway promoting the formation of STAT1-IRF9 transcription factor complex. Following this, OSM increased the expression of various genes with known functions in innate and adaptive immunity, which was higher expression in patients with CHB in immune clearance phase than in immune tolerance phase (data from GEO: GSE65359). Interferon-induced transmembrane protein 1, one of the most differentially expressed genes, was identified as an HBV restriction factor involved in OSM-mediated anti-HBV effect. In vivo, we also found OSM significantly inhibited HBV replication and induced expression of antiviral effector interferon-induced transmembrane protein 1. CONCLUSIONS: Our study shows that OSM remodels the immune response against HBV and exerts potent anti-HBV activity, supporting its further development as a potential therapy for treating CHB.

MicroRNA-363-3p inhibits colorectal cancer progression by targeting interferon-induced transmembrane protein 1.

BACKGROUND: The molecular mechanisms of colorectal cancer development and progression are far from being elucidated. AIM: To investigate the role of microRNA-363-3p (miR-363-3p) in the progression of colorectal cancer. METHODS: Real-time polymerase chain reaction was performed to detect miRNA expression in human colorectal cancer tissues and paired normal colorectal tissues. PITA 6 was utilized to predict the targets of miR-363-3p. Dual-luciferase reporter system was used to validate the target of miR-363-3p. Plate colony formation assay and wound-healing assay were performed to evaluate cancer cells' clonogenic survival ability and migration ability, respectively. Cell proliferation was examined by cell counting kit-8 assay. Immunohistochemical staining was used to determine the expression level of interferon-induced transmembrane protein 1 (IFITM1) in colorectal cancer tissues and adjacent tissues. The TCGA and GTEx databases were used to compare the expression levels of IFITM1 mRNA in colorectal cancer tissues and normal colorectal tissues and analyze the correlation between the expression levels of IFITM1 mRNA and overall survival and disease-free survival of patients. A colorectal cancer cell line with a deficiency of IFITM1 was constructed, and the regulation effect of IFITM1 on the clonogenic growth of colorectal cancer cells was clarified. RESULTS: MiR-363-3p was decreased in colorectal cancer tissues compared to normal colorectal tissues. IFITM1 was characterized as a direct target of miR-363-3p. Overexpression of miR-363-3p led to decreased clonogenic survival, proliferation, and migration of colorectal cancer cells, which could be reversed by forced IFITM1 expression. CONCLUSION: MiR-363-3p can constrain clonogenic survival, proliferation, and migration of colorectal cancer cells via targeting IFITM1.

MiR-504-3p Has Tumor-Suppressing Activity and Decreases IFITM1 Expression in Non-Small Cell Lung Cancer Cells.

Objective: To analyze the impact of expression of miR-504-3p on the proliferation, migration, cell cycle transit and rate of apoptosis of NSCLC cells and explore the underlying mechanisms. Methods: The Cancer Genome Atlas (TCGA) database was used to compare the expression levels of miR-504 between NSCLC tissues and normal lung tissues. NSCLC cells were transfected with lentiviral vectors that either overexpressed or knocked down miR-504-3p to evaluate its effects on NSCLC biological behavior. Quantitative Real Time Polymerase Chain Reaction was used to measure the levels of miR-504-3p and Interferon-Induced Transmembrane Protein 1 (IFITM1). A luciferase reporter array was used to reveal whether miR-504-3p directly targets IFITM1. Results: The expression of miR-504 was significantly down-regulated in lung cancer tissues compared to normal lung tissues. Overexpression of miR-504-3p in NSCLC cell lines inhibited cell proliferation, migration and promoted cell apoptosis. Meanwhile, changes in the expression level of miR-504-3p had no significant effect on NSCLC cell cycle progression. Moreover, over-expressed miR-504-3p following its transfection significantly decreased the expression of IFITM1 in NSCLC cell lines and suppressed the activity of the luciferase reporter containing wild type but not mutant IFITM1 3' -UTR. Conclusion: miR-504-3p inhibits cell proliferation and migration and promotes cell apoptosis in NSCLC cells. MiR-504-3p decreases IFITM1 expression in NSCLC cells, which may be a potential mechanism of its tumor-suppressive functions in NSCLC.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Epigenetic regulation of IFITM1 expression in lipopolysaccharide-stimulated human mesenchymal stromal cells.

BACKGROUND: Toll-like receptor 4 (TLR4) ligands such as lipopolysaccharide (LPS) activate immunomodulatory functions and the migration of human mesenchymal stromal cells (hMSCs). Here, we study the migration-related gene expression of LPS-stimulated hMSCs and the role and regulation of one of the upregulated genes, encoding the interferon-induced transmembrane protein 1 (IFITM1). METHODS: Gene expression profiles were determined by whole-transcriptome analysis (RNA-seq) and quantitative real-time PCR (qRT-PCR). Bioinformatics approaches were used to perform network and pathway analyses. The cell migration-related genes were identified with an in vitro wound healing assay. RNA interference (RNAi) was used to suppress the IFITM1 gene expression. The IFITM1 gene enhancer was analyzed by chromatin immunoprecipitation (ChIP) sequencing, ChIP-to-PCR, luciferase reporter assays, and qRT-PCR for enhancer RNAs (eRNAs). RESULTS: RNA-seq confirmed IFITM1 as an LPS-stimulated gene, and RNAi demonstrated its importance for the LPS-stimulated migration. LPS treatment increased the eRNA expression in enhancer region R2 (2 kb upstream) of the IFITM1 gene and enriched R2 for H3K27ac. Bioinformatics implicated the transcription factors NF-κB and IRF1, ChIP assays revealed their binding to R2, and chemical inhibition of NF-κB and RNAi directed against IRF1 prevented R2 eRNA and IFITM1 gene expression. CONCLUSIONS: Increased expression of the IFITM1 gene is required for LPS-stimulated hMSC migration. We described several underlying changes in the IFITM1 gene enhancer, most notably the NF-κB-mediated activation of enhancer region R2.

Interferon-induced transmembrane protein 1 (IFITM1) is essential for progression of laryngeal squamous cell carcinoma in an Osteopontin/NF-κB-dependent manner.

OBJECTIVE: To assess the expression levels of IFITM1 in human tissue samples and laryngeal squamous cell carcinoma (LSCC) cells, and to explore the potential mechanisms of IFITM1 in LSCC progression. METHODS: Quantitative PCR and immunohistochemical (IHC) assays were performed to detect IFITM1 expression in 62 LSCC tissues and corresponding normal tissues. We further detected the effects of IFITM1 on the proliferation, migration and invasion of LSCC cells and NF-κB signaling pathway through colony formation assay, wound healing assay and transwell assay, respectively. RESULTS: We demonstrated the possible involvement of IFITM1 in the progression of LSCC. We found the upregulated expression of IFITM1 in human LSCC tissues and cells, and analyzed the correlations between IFITM1 expression and osteopontin. Our data further confirmed that IFITM1 affected cell proliferation, migration, and invasion of LSCC cells via the regulation of NF-κB signaling pathway. CONCLUSIONS: We investigated the potential involvement of IFITM1 in the progression of LSCC, and therefore confirmed that IFITM1 was a potential therapeutic target for LSCC.

Prognostic significance of IFITM1 expression and correlation with microvessel density and epithelial-mesenchymal transition signature in lung adenocarcinoma.

We evaluated the relationship between interferon-induced transmembrane protein 1 (IFITM1) expression, epithelial-mesenchymal transition (EMT) signature and angiogenesis in lung adenocarcinoma. Additionally, we examined prognostic significance of IFITM1 according to pTNM stage to confirm that IFITM1 can serve as a complement to the pTNM stage. A total of 141 lung adenocarcinoma specimens were evaluated retrospectively by immunohistochemical staining for IFITM1, EMT markers (e-cadherin, ß-catenin, and vimentin), and CD31 to measure microvessel density. IFITM1was expressed in 46.8% of the specimens. IFITM1 expression was significantly correlated with increased microvessel density (P = 0.048). However, IFITM1 expression was not associated with three EMT markers. In a multivariate analysis, IFITM1 was an independent prognostic factor for overall survival in a multivariate analysis (hazard ratio: 2.59, P = 0.01). Online database with data from 720 lung adenocarcinoma patients also revealed a negative prognostic significance of IFITM1 (P < 0.001). Furthermore, high IFITM1 expression was significantly correlated with decreased OS rates in each pTNM stage. IFITM1 is significantly correlated with angiogenesis and it may be used as a useful additional prognostic marker to aid pTNM classification.

Human Hepatitis B Virus Core Protein Inhibits IFNα-Induced IFITM1 Expression by Interacting with BAF200.

Human hepatitis B virus core protein (HBc) is a structural protein of the hepatitis B virus (HBV) and contributes to HBV regulation of host-cell transcription. However, the mechanisms of transcriptional regulation remain poorly characterized. To dissect the function of HBc, a yeast two-hybrid was performed to identify HBc-binding proteins, and the C-terminal of BRG1/hBRM-associated factors 200 (BAF200C) was identified. Then, the existence of HBc interactions with BAF200C and full-length BAF200 was confirmed via co-immunoprecipitation assays in 293T, HepG2 and HepG2-NTCP cells. Furthermore, we show that the binding between HBc and BAF200 was of vital importance to HBc mediated downregulation of interferon-induced transmembrane protein 1 (IFITM1) expression, and the mechanisms for the downregulation were disclosed as follows. Basal level of IFITM1 expression depends on BAF200, rather than the JAK-STAT1 pathway. The interaction of HBc with BAF200 disturbs the stability of the polybromo-associated BAF (PBAF) complex and results in the suppression of IFTM1 transcription. Finally, the antiviral effects of IFITM1 on cell proliferation and HBV replication were found to be partially restored when HBc was co-transfected with BAF200. Collectively, our findings indicate that HBc plays a role in HBV resistance against the antiviral activities of IFNα, providing details about HBV evasion of host innate immunity.