Interferon-induced Transmembrane Protein 1 Expression Research Articles

IFITM1 expression is crucial to gammaherpesvirus infection, in vivo

The oncogenic gammaherpesviruses, Epstein–Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV), are etiologically associated with a variety of human cancers, including Burkitt’s lymphoma (BL), Hodgkin lymphoma (HL), Kaposi’s sarcoma (KS), and primary effusion lymphoma (PEL). Recently, we demonstrated KSHV infection of B- and endothelial cells to significantly upregulate the expression of interferon induced transmembrane protein 1 (IFITM1) which in turn enhances virus entry. This is an extension of the above study. In here, we determined EBV infection of cells to trigger IFITM1 expression, in vitro. Silencing IFITM1 expression using siRNA specifically lowered gammaherpesvirus infection of cells at a post binding stage of entry. A natural model system to explore the effect of IFITM1 on gammaherpesvirus infection in vivo is infection of BALB/c mice with murine gammaherpesvirus 68 (MHV-68). Priming mice with siRNA specific to IFITM1 significantly lowered MHV-68 titers in the lung specimens compared to priming with (NS)siRNA or PBS. MHV-68 titers were monitored by plaque assay and qPCR. Taken together, for the first time, this study provides insight into the critical role of IFITM1 to promoting in vivo gammaherpesvirus infections.

Distribution of Ifitm3 in Yellow-Feathered Broilers and Inhibition of Avian Reovirus Multiplication by Ifitm3

ABSTRACT This study was carried to express the interferon-induced transmembrane protein 3 (IFITM3) in vitro and examine its function in inhibition of avian reovirus (ARV) replication. The recombinant prokaryotic vector expressing yellow-feathered broiler IFITM3 was successfully constructed, and the recombinant protein was expressed in competent Escherichia coli BL21 cells. New Zealand white rabbits were immunized with the purified recombinant protein to prepare a polyclonal antibody, with a titer of 1:128,000. Immunohistochemistry, reverse transcription-PCR, and real-time fluorescence quantitative PCR showed that IFITM3 was distributed in the yellow-feathered broiler immune organs, and the expression of IFITM3 in bursa of Fabricius was more than in spleen and thymus. It was found that in the thymus, spleen and bursa of Fabricius the mRNA expression levels of IFNα and IFITM3 were significantly induced after ARV infection. And it was also certified in the chicken embryo fibroblasts (CEFs) which infected with ARV. Then the IFNα was added into the cell culture medium before CEFs were infected with ARV. The results indicated that the mRNA of IFITM3 expression was significantly increased and ARV multiplication was significantly inhibited. And when the expression of IFITM3 was knocked down by siRNA-IFITM3, the expression of IFITM3 was significantly reduced, but the ARV multiplication was significantly increased, which indicated that IFITM3 protein could inhibit the ARV replication.

Zoledronate suppresses VEGF‑induced capillary tube formation and inhibits expression of interferon‑induced transmembrane protein‑1 in human umbilical vein endothelial cells.

Interferon‑induced transmembrane protein1 (IFITM1) is a member of the interferon‑induced transmembrane protein family and has recently been identified as a novel protein participant in angiogenesis. Zoledronate(ZON), a nitrogen‑containing bisphosphonate, is widely used in the treatment of osteoporosis and to prevent bone metastases of certain cancer types. However, the association between ZON and IFITM1 has remained elusive. The present study investigated the effect of ZON on the expression of IFITM1 during vascular endothelial growth factor (VEGF)‑induced capillary tube formation in human umbilical vein endothelial cells. It was observed that cell proliferation and VEGF‑induced tube formation were significantly inhibited by treatment with 10µM ZON. The expression of IFITM1 increased during VEGF‑induced tube formation. However, the VEGF‑induced increase in IFITM1 expression exhibited a dose‑ and time‑dependent decrease with ZON treatment at the mRNA and protein level. Furthermore, matrix metalloproteinase‑9 activation was markedly decreased by ZON treatment. These results suggest that induction of IFITM1 expression may be involved in the anti‑angiogenic activity of ZON.

Expression and Cellular Localization of IFITM1 in Normal and Injured Rat Spinal Cords.

Interferon-induced transmembrane protein 1 (IFITM1) is a member of the IFITM family that is associated with some acute-phase cytokine-stimulated response. Recently, we demonstrated that IFITM1 was significantly upregulated in the injured spinal cords at the mRNA level. However, its expression and cellular localization at the protein level is still unclear. Here, a rat model of spinal cord injury (SCI) was performed to investigate the spatio-temporal expression of IFITM1 after SCI. IFITM1 mRNA and protein were assessed by quantitative reverse transcription-PCR and western blot, respectively. IHC was used to identify its cellular localization. We revealed that IFITM1 could be found in sham-opened spinal cords and gradually increased after SCI. It reached peak at 7 and 14 days postinjury (dpi) and still maintained at a relatively higher level at 28 dpi. IHC showed that IFITM1 expressed in GFAP+ and APC+ cells in sham-opened spinal cords. After SCI, in addition to the above-mentioned cells, it could also be found in CD45+ and CD68+ cells, and its expression in CD45+, CD68+, and GFAP+ cells was increased significantly. These results demonstrate that IFITM1 is mainly expressed in astrocytes and oligodendroglia in normal spinal cords, and could rapidly increase in infiltrated leukocytes, activated microglia, and astrocytes after SCI.

MiRNA-36 inhibits KSHV, EBV, HSV-2 infection of cells via stifling expression of interferon induced transmembrane protein 1 (IFITM1)

Kaposi’s sarcoma-associated herpesvirus (KSHV) is etiologically associated with all forms of Kaposi’s sarcoma worldwide. Little is currently known about the role of microRNAs (miRNAs) in KSHV entry. We recently demonstrated that KSHV induces a plethora of host cell miRNAs during the early stages of infection. In this study, we show the ability of host cell novel miR-36 to specifically inhibit KSHV-induced expression of interferon induced transmembrane protein 1 (IFITM1) to limit virus infection of cells. Transfecting cells with miR-36 mimic specifically lowered IFITM1 expression and thereby significantly dampening KSHV infection. In contrast, inhibition of miR-36 using miR-36 inhibitor had the direct opposite effect on KSHV infection of cells, allowing enhanced viral infection of cells. The effect of miR-36 on KSHV infection of cells was at a post-binding stage of virus entry. The highlight of this work was in deciphering a common theme in the ability of miR-36 to regulate infection of closely related DNA viruses: KSHV, Epstein-Barr virus (EBV), and herpes simplexvirus-2 (HSV-2). Taken together, we report for the first time the ability of host cell miRNA to regulate internalization of KSHV, EBV, and HSV-2 in hematopoietic and endothelial cells.

Down-regulation of IFITM1 and its growth inhibitory role in cervical squamous cell carcinoma

BackgroundCervical cancer is a major cause of death in women worldwide. Interferon-induced transmembrane protein 1 (IFITM1) is involved in antivirus defense, cell adhesion, and carcinogenesis in different tissues. However, the role of IFITM1 gene in cervical squamous cell cancer is unclear.MethodsTo explore the role of IFITM1 in carcinogenesis of cervical cancer, we investigated the expression of IFITM1 gene in cervical squamous cell carcinoma. IFITM1 mRNA level was measured by real-time quantitative RT-PCR in cervical cancer tissues and their adjacent normal tissues. IFITM1 protein level was measured by immunohistochemistry. Methylation in the IFITM1 gene promoter was detected by methylation-specific PCR. We then transfected HeLa cells with IFITM1 expression vector or control vector. IFITM1 expression was examined; cell migration and invasion were analyzed by wound healing assay and matrigel-coated transwell migration assays, respectively. HeLa cell proliferation was measured by cell counting kit-8 assay and cell cycle analysis. Cell apoptosis was analyzed by Annexin V/propidium iodide double staining assay.ResultsThe difference in IFITM1 protein expression between samples from chronic cervicitis and cervical carcinoma was statistically significant (P < 0.01). Ki-67 and PCNA protein expression levels were significantly higher in cervical cancer tissues than in their corresponding cervicitis tissues (P < 0.05 and P < 0.001, respectively). IFITM1 mRNA level was significantly lower in cervical cancer tissues than in normal cervical tissues (P < 0.05). Methylation of the IFITM1 gene promoter was significantly higher in cervical cancer than in normal cervical tissues (P < 0.05). Transfection of the IFITM1 pcDNA3.1 construct decreased cell migration and invasion of HeLa cells, inhibited cell proliferation, and increased cell apoptosis.ConclusionIFITM1 gene expression may reduce the proliferation, migration, and invasion of cervical squamous cancer cells.

Abstract 3848: The clinical relevance of targeting IFITM1 in three distinct subtypes of aggressive breast cancer

Abstract Breast cancer can be separated into estrogen receptor/ progesterone receptor positive (ER+/PR+), Her2/neu positive (HER2+), and triple negative (ER-/PR-/HER2-) subtypes. Additionally, breast cancer may manifest clinically as either non-inflammatory or inflammatory disease. Inflammatory breast cancer is a highly lethal subtype of breast carcinoma that may express any of the three aforementioned receptors. Our laboratory has found that a novel marker, interferon-induced transmembrane protein 1 (IFITM1), is overexpressed in multiple subtypes of breast cancer and its overexpression is associated with poor overall survival as well as resistance to endocrine therapy, chemotherapy, and radiation. IFITM1 is a type 1 interferon (IFN) stimulated gene that is not expressed in normal breast tissue, and is only induced upon IFN exposure. In this study, we conducted immunohistochemical staining for IFITM1 in 94 ER+ human breast tumor samples and discovered that high IFITM1 expression was associated with increased clinical stage and resistance to endocrine therapy. Elevated expression of IFITM1 was also detected in human triple negative breast cancer (TNBC) samples as compared to normal breast tissue. Notably, this overexpression was most pronounced in TNBC tumors from African American (AA) patients. We screened a panel of nine cell lines that represent all major subtypes of breast cancer and found IFITM1 overexpression in ER+ aromatase inhibitor-resistant MCF-7:5C, AA-derived triple negative MDA-MB-468 and MDA-MB-157, and triple negative inflammatory SUM149 cells. We used inducible shRNA and CRSPR/Cas9 respectively, to investigate the effect of IFITM1 knockdown on MCF-7:5C and SUM149 xenograft tumors using two in vivo breast cancer models. The orthotopic (mammary fat pad) model evaluated tumor proliferation, and the mammary intraductal (MIND) model assessed tumor cell invasion out of the milk duct. We found that loss of IFITM1 in MCF-7:5C and SUM149 cells significantly inhibited their ability to form tumors in vivo and it completely blocked their ability to invade out of the milk duct. Additional studies indicated that hyper-activated JAK/STAT signaling was responsible for driving IFITM1 expression in MCF-7:5C, MDA-MB-468, and SUM149 cells. Furthermore, we demonstrated that JAK/STAT activation can be targeted in vivo with the FDA approved JAK inhibitor ruxolitinib (Jakafi™). Oral treatment of mice with 50 μg/g bodyweight ruxolitinib reduced IFITM1 expression and inhibited the growth of both MCF-7:5C and MDA-MB-468 tumors by loss of STAT1/2 phosphorylation. The overexpression of IFITM1 in three distinct breast cancer subtypes indicates that IFITM1 may be a targetable marker of aggressive disease in general and may open the door for novel therapies of treatment-refractory breast cancer. Citation Format: Asona Lui, Joshua Ogony, Jordan Marquess, Eric Geanes, William Jewell, Ikbale El Ayachi, Gustav Miranda-Carboni, Ossama Tawfik, Joan Lewis-Wambi. The clinical relevance of targeting IFITM1 in three distinct subtypes of aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3848. doi:10.1158/1538-7445.AM2017-3848

Evaluation of IFITM3 rs12252 Association With Severe Pediatric Influenza Infection.

Interferon-induced transmembrane protein 3 (IFITM3) restricts endocytic fusion of influenza virus. IFITM3 rs12252_C, a putative alternate splice site, has been associated with influenza severity in adults. IFITM3 has not been evaluated in pediatric influenza. The Pediatric Influenza (PICFLU) study enrolled children with suspected influenza infection across 38 pediatric intensive care units during November 2008 to April 2016. IFITM3 was sequenced in patients and parents were genotyped for specific variants for family-based association testing. rs12252 was genotyped in 54 African-American pediatric outpatients with influenza (FLU09), included in the population-based comparisons with 1000 genomes. Splice site analysis of rs12252_C was performed using PICFLU and FLU09 patient RNA. In PICFLU, 358 children had influenza infection. We identified 22 rs12252_C homozygotes in 185 white non-Hispanic children. rs12252_C was not associated with influenza infection in population or family-based analyses. We did not identify the Δ21 IFITM3 isoform in RNAseq data. The rs12252 genotype was not associated with IFITM3 expression levels, nor with critical illness severity. No novel rare IFITM3 functional variants were identified. rs12252 was not associated with susceptibility to influenza-related critical illness in children or with critical illness severity. Our data also do not support it being a splice site.

IFITM1 suppression blocks proliferation and invasion of aromatase inhibitor-resistant breast cancer in vivo by JAK/STAT-mediated induction of p21

Interferon induced transmembrane protein 1 (IFITM1) belongs to a family of interferon stimulated genes (ISGs) that is associated with tumor progression and DNA damage resistance; however, its role in endocrine resistance is not known. Here, we correlate IFITM1 expression with clinical stage and poor response to endocrine therapy in a tissue microarray consisting of 94 estrogen receptor (ER)-positive breast tumors. IFITM1 overexpression is confirmed in the AI-resistant MCF-7:5C cell line and not found in AI-sensitive MCF-7 cells. In this study, the orthotopic (mammary fat pad) and mouse mammary intraductal (MIND) models of breast cancer are used to assess tumor growth and invasion in vivo. Lentivirus-mediated shRNA knockdown of IFITM1 in AI-resistant MCF-7:5C cells diminished tumor growth and invasion and induced cell death, whereas overexpression of IFITM1 in wild-type MCF-7 cells promoted estrogen-independent growth and enhanced their aggressive phenotype. Mechanistic studies indicated that loss of IFITM1 in MCF-7:5C cells markedly increased p21 transcription, expression and nuclear localization which was mediated by JAK/STAT activation. These findings suggest IFITM1 overexpression contributes to breast cancer progression and that targeting IFITM1 may be therapeutically beneficial to patients with endocrine-resistant disease.

Interferon-induced transmembrane protein 1 (IFITM1) is required for the progression of colorectal cancer.

Interferon-induced transmembrane protein 1 (IFITM1) has been shown to be implicated in multiple cancers, yet little is known about biological significance of IFITM1 in colorectal cancer. Here, we show that IFITM1 is highly expressed in metastatic colorectal cancer cell lines as well as colorectal patient-derived tumor samples, and its expression is associated with a poor prognosis of the disease. Also, IFITM1 depletion resulted in a significant reduction in the mobility of cancer cell lines, whereas ectopic expression of IFITM1 promoted the migration of cancer cells. Epithelial-mesenchymal transition (EMT) signature was dysregulated by both loss and gain of function of IFITM1, which was partially reverted by Caveolin-1 (CAV1). Therefore, these results suggest that IFITM1 may be a prognostic marker and an attractive target to achieve better therapeutic outcomes in colorectal cancer.

Mitochondrial interferon-induced transmembrane protein-1 is a critical regulator of cell death in MPRO cells

Interferon-induced transmembrane protein 1 (IFITM1) is a transmembrane protein that is an essential mediator of interferon-induced anti-proliferation. Identifying the functional role of IFITM1 has been difficult because of high expression of the protein induces cell death. In this study, we showed that IFITM1 is localized to the mitochondria and that its expression is related to mitochondrial reactive oxygen species (mROS). Overexpression of IFITM1 resulted in a wrinkled and ruptured cell morphology, and subcellular fractionation revealed IFITM1 expression in the mitochondria and the plasma membrane. Interestingly, IFITM1 was expressed at high levels in the mitochondria of MPRO cells, but only at low levels in the plasma membrane. A mitochondrial staining assay confirmed that IFITM1 was localized to the mitochondria in MPRO cells. In addition, IFITM1 expression was significantly increased by treatment with H2O2, arachidonic acid, and dithiothreitol. These findings suggest that IFITM1 expression is not restricted to the membrane, but is present in the mitochondria as well. Furthermore, mitochondrial IFITM1 expression is regulated by mROS.

Abstract 2918: Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells

Abstract Aromatase inhibitors (AIs) successfully treat many estrogen receptor expressing breast cancers by depleting circulating estrogen levels. Unfortunately, many patients eventually develop resistance to this treatment. In order to elucidate the mechanism(s) of AI-resistance, our lab has developed an AI-resistant breast cancer cell line MCF-7:5C which was clonally derived from parental MCF-7 cells following long-term estrogen deprivation. Unlike MCF-7 cells, the AI-resistant MCF-7:5C cell line grows robustly in the absence of estrogen and is highly aggressive. We have previously reported that interferon induced transmembrane protein 1 (IFITM1) is overexpressed in MCF-7:5C cells and in AI-resistant breast tumors. Additionally, we have shown that loss of IFITM1 leads to cell death. What remains to be addressed is how IFITM1 expression is regulated in AI-resistant breast cancer cells. IFITM1 is a type 1 interferon (IFN) stimulated gene which is not expressed in normal tissue and is only induced by the type1 IFNs (IFNα/β). In previous studies we have found that MCF-7:5C cells produce elevated levels of IFNα, which binds to the type 1 IFN receptor, IFNAR, and induces JAK/STAT signaling, resulting in the overexpression of IFITM1. In this study, we further characterized the mechanisms of IFITM1 overexpression in AI-resistant MCF-7:5C cells. We have found that mucin 1 (MUC1) associates with STAT1 and stabilizes its phosphorylated form, thereby enhancing IFITM1 expression. This interaction is disrupted by treatment with the JAK inhibitor, Ruxolitinib. MUC1 is a transmembrane O-glycosylated protein that is overexpressed in 90% of breast cancers and known to promote the survival of the epithelium through formation of mucous, interaction with transcription factors and inhibition of apoptosis. A luciferase reporter determined that MUC1 contributes directly to IFITIM1 transcription. MUC1 expression is hormonally controlled and is normally enhanced by estrogen stimulation. In this study we found that MUC1 expression is dysregulated in AI-resistant MCF-7:5C cells and is instead reduced by estrogen stimulation. In contrast with the AI-sensitive MCF-7 cells, western blot and immunofluorescent staining showed that MUC1 expression was enhanced by treatment with the pure anti-estrogen, fulvestrant. Additionally, Annexin V/PI staining and PARP cleavage indicated that loss of MUC1 expression induced cell death in MCF-7:5C cells. We found that treatment with fulvestrant protects MCF-7:5C cells from this phenomenon, indicating that loss of MUC1 induces apoptosis in an ERα dependent manner. MCF-7:5C cells are sensitive to apoptosis following exposure to estrogen, which can be enhanced by loss of IFITM1 expression. We report that loss of MUC1 expression similarly enhances estrogen-induced apoptosis, suggesting that the communication between MUC1 and STAT1 also influences estrogen signaling in AI-resistant breast cancer. Citation Format: Asona Lui, Joan Lewis-Wambi. Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2918.

IFITM1 Is Superior to CD10 as a Marker of Endometrial Stroma in the Evaluation of Myometrial Invasion by Endometrioid Adenocarcinoma.

Background: Distinguishing myometrial invasion from adenomyosis involvement is important for staging of endometrial endometrioid adenocarcinoma. We aimed to compare CD10, which has limited value in this scenario, with interferon-induced transmembrane protein 1 (IFITM1), a recently described sensitive and specific marker of endometrial stroma. Methods: We reviewed 25 hysterectomies containing endometrial endometrioid adenocarcinoma and adenomyosis. Tumor areas were classified as unequivocally myoinvasive or unequivocally noninvasive. Foci equivocal for invasion were also recorded. Immunohistochemistry for IFITM1 and CD10 was performed and scored in terms of intensity and distribution and classified as negative or positive. Results: Unlike CD10, IFITM1 staining showed significant differences in mean intensity (P < .0001) and distribution (P < .0001) between invasive vs noninvasive areas. Sixteen (84.2%) invasive and 34 (97.1%) noninvasive areas were positive for CD10 (P = .22). In contrast, none of the invasive vs 25 (71.4%) noninvasive areas were positive for IFITM1 (P < .0001). IFITM1 had 71.4% sensitivity and 100% specificity in detecting stroma surrounding endometrioid adenocarcinoma, hence excluding myoinvasion. Eleven (45.8%) of 24 foci designated as equivocal stained with IFITM1. Conclusions: Compared with CD10, IFITM1 has superior performance distinguishing endometrial stroma of adenomyosis from mesenchyma surrounding invasive endometrial adenocarcinoma. IFITM1 expression is highly predictive of the absence of invasion and may be valuable in cases in which determining myoinvasion has staging implications.

Interferon-induced transmembrane protein 1 (IFITM1) overexpression enhances the aggressive phenotype of SUM149 inflammatory breast cancer cells in a signal transducer and activator of transcription 2 (STAT2)-dependent manner.

BackgroundInflammatory breast cancer (IBC) is a very aggressive and lethal subtype of breast cancer that accounts for about 4 % of all breast cancers diagnosed in the United States. Despite the efforts of several investigators to identify the molecular factors driving the aggressive phenotype of IBC, a great deal is still unknown about the molecular underpinnings of the disease. In the present study, we investigated the role of interferon-induced transmembrane protein 1 (IFITM1), a well-known interferon-stimulated gene (ISG), in promoting the aggressiveness of SUM149 IBC cells.MethodsWestern blot and real-time polymerase chain reaction analyses were performed to assess the protein and messenger RNA (mRNA) levels of IFITM1 and other ISGs in three IBC cell lines: SUM149, MDA-IBC-3, and SUM190. IFITM1 expression and cellular localization were assessed by using immunofluorescence, while the tumorigenic potential was assessed by performing cell migration, invasion, and colony formation assays. Small interfering RNA and short hairpin RNA knockdowns, enzyme-linked immunosorbent assays, and luciferase assays were performed to determine the functional significance of IFITM1 and signal transducers and activators of transcription 1 and 2 (STAT1/2) in SUM149 cells.ResultsWe found that IFITM1 was constitutively overexpressed at the mRNA and protein levels in triple-negative SUM149 IBC cells, but that it was not expressed in SUM190 and MDA-IBC-3 IBC cells, and that suppression of IFITM1 or blockade of the IFNα signaling pathway significantly reduced the aggressive phenotype of SUM149 cells. Additionally, we found that knockdown of STAT2 abolished IFITM1 expression and IFITM1 promoter activity in SUM149 cells and that loss of STAT2 significantly inhibited the ability of SUM149 cells to proliferate, migrate, invade, and form 2-D colonies. Notably, we found that STAT2-mediated activation of IFITM1 was particularly dependent on the chromatin remodeler brahma-related gene 1 (BRG1), which was significantly elevated in SUM149 cells compared with SUM190 and MDA-IBC-3 cells.ConclusionsThese findings indicate that overexpression of IFITM1 enhances the aggressive phenotype of triple-negative SUM149 IBC cells and that this effect is dependent on STAT2/BRG1 interaction. Further studies are necessary to explore the potential of IFITM1 as a novel therapeutic target and prognostic marker for some subtypes of IBCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0683-7) contains supplementary material, which is available to authorized users.

IFITM3 (CD225) Links the B Cell Antigen CD19 to PI3K-AKT Signaling in Human ALL Cells

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21. Recent immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013). However, in some cases, CART19 treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Therefore, we studied factors that regulate CD19 surface expression on human pre-B ALL cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and activation of checkpoint molecules p53 and p21, reduction of BCL2 and BCLXL levels and increased propensity to apoptosis. Reconstitution of IFITM3 reversed these changes.Conversely, forced expression of IFITM3 in patient-derived pre-B ALL cells increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Although human IFITM1 has known as a component of B cell receptor complex, co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT. In addition, agonistic antibodies against IFITM3/CD225 trigger CD19/PI3K-AKT signaling, which caused massively increased proliferation of pre-B ALL cells. Interestingly, agonistic antibodies against CD19 had no effect on the proliferation of pre-B ALL cells despite their ability to activate CD19/PI3K-AKT signaling. These findings indicate a specific role of IFITM3 in regulating CD19/PI3K-AKT signaling in malignant pre-B ALL cells compared to their normal pre-B cell counterparts.Conclusion: These findings identify novel role of the IFITM3 (CD255) surface receptor in regulating CD19 phosphorylation and CD19-Y513 mediated PI3K-AKT signaling in pre-B ALL cells. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K-AKT in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Respiratory DC Use IFITM3 to Avoid Direct Viral Infection and Safeguard Virus-Specific CD8+ T Cell Priming.

Respiratory dendritic cells (DC) play a pivotal role in the initiation of adaptive immune responses to influenza virus. To do this, respiratory DCs must ferry viral antigen from the lung to the draining lymph node without becoming infected and perishing en route. We show that respiratory DCs up-regulate the expression of the antiviral molecule, interferon-induced transmembrane protein 3 (IFITM3) in response to influenza virus infection, in a manner dependent on type I interferon signaling and the transcription factors IRF7 and IRF3. Failure of respiratory DCs to up-regulate IFITM3 following influenza virus infection resulted in impaired trafficking to the draining LN and consequently in impaired priming of an influenza-specific CD8+ T cell response. The impaired trafficking of IFITM3-deficient DC correlated with an increased susceptibility of these DC to influenza virus infection. This work shows that the expression of IFITM3 protects respiratory DCs from influenza virus infection, permitting migration from lung to LN and optimal priming of a virus specific T-cell response.

IFITM1 promotes the metastasis of human colorectal cancer via CAV-1

Interferon-induced transmembrane protein 1 (IFITM1) is one of the interferon-induced transmembrane protein family members. In this study, we reported that the elevated IFITM1 expression in human colorectal cancer (CRC) significantly correlated with CRC lymph node and distance metastasis as well as a more advanced clinical stage. Importantly, elevated IFITM1 expression is an independent prognostic factor for poor survival. To investigate the molecular mechanisms, we showed that over-expression of IFITM1 in CRC cells promoted, whereas knockdown of IFITM1 expression inhibited, cell migration/invasion and tumorigenicity in vitro. Furthermore, we identified Caveolin-1 (CAV1) as a downstream target of IFITM1-induced cell invasion, as knockdown of CAV1 abrogated siIFITM1 mediated inhibition of cell invasion in CRC cells. In addition, in a CRC cohort of 229 patients, the expression of IFITM1 inversely correlated with the expression of CAV1. These results suggested that IFITM1 promotes the aggressiveness of CRC cells, and it is a potential prognostic marker and therapeutic target for CRC.

Abstract 3560: IFITM1 overexpression enhances the aggressive phenotype of inflammatory breast cancer in a STAT2-dependent manner

Abstract Inflammatory breast cancer (IBC) is a highly metastatic, aggressive, and fatal form of breast cancer that accounts for about 1 to 5% of all breast cancers diagnosed in the United States. At present, the molecular mechanisms that underlie the aggressive phenotype of IBC are not known. Interferon induced transmembrane protein1 (IFITM1) is a member of interferon stimulated genes (ISGs), whose overexpression has been linked to several malignancies, but its role in inflammatory breast cancer is not known. The primary role of IFITM1 is to stop the spread of viral pathogens in the host's cells, but its constitutive overexpression has been shown to enhance malignancy. IFITM1 expression is known to be regulated by type 1 interferon through activation of the canonical JAK-STAT pathway; however, recent studies suggest that non-canonical signaling pathways involving STAT2 homodimers can also regulate IFITM1 expression. In the present study, we determined whether IFITM1 plays a critical role in driving the aggressive phenotype of three IBC cell lines; SUM149; MDA-IBC-3, and SUM190. Western blot and RT-PCR analyses indicated that IFITM1 was constitutively overexpressed at the protein (∼5- to 10-fold) and mRNA (∼4-fold) level, respectively, in SUM149 and MDA-IBC-3 cells but not expressed in non-IBC MCF-7 luminal breast cancer cells, and that its overexpression strongly correlated with increased proliferation, migration and invasion, and enhanced colony formation. Notably, the aggressive phenotype of SUM149 and MDA-IBC-3 cells was completely reversed by siRNA knockdown of IFITM1. Specifically, we found that suppression of IFITM1 significantly reduced the ability of SUM149 and MDA-IBC-3 cells to proliferate, migrate and invade, and form colonies. Furthermore, we found that IFITM1 expression and promoter activity were completely abolished due to STAT2 knockdown and that BRG1 (Brahma-related-gene 1) was crucial for the expression of IFITM1 and STAT2 in the IBC cells. Moreover, we found that knockdown of BRG1; a member of the mammalian chromatin-remodeling complex markedly reduced IFITM1 and STAT2 proteins, but not STAT1, in SUM149 and MDA-IBC-3 cells, thus supporting a critical role for STAT2/BRG1 in the regulation of IFITM1 expression in these cells. Taken together, this data suggests that constitutive overexpression of IFITM1 in inflammatory breast cancer cells enhances the aggressive phenotype of these cells, and that STAT2 and BRG1 play pivotal roles in driving IFITM1 overexpression. Additionally, this data suggests that IFITM1 may be a suitable candidate as a novel therapeutic target and a potential prognostic maker for inflammatory breast cancer. Citation Format: Joshua Were Ogony, Joan Lewis-Wambi. IFITM1 overexpression enhances the aggressive phenotype of inflammatory breast cancer in a STAT2-dependent manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3560. doi:10.1158/1538-7445.AM2015-3560

Mechanism and biological significance of the overexpression of IFITM3 in gastric cancer

Interferon‑induced transmembrane protein 3 (IFITM3) has been recently identified as a potential molecular marker. IFITM3 has been reported to be upregulated in various human diseases, including colon and breast cancer, astrocytoma, as well as ulcerative colitis. However, the clinical significance and underlying mechanisms of IFITM3 dysregulated expression in gastric cancer (GC) remain to be determined. The present study aimed to evaluate the expression of IFITM3 in human gastric tumor cells and specimens and investigate the effects of IFITM3 knockdown in the regulation of GC growth and its potential mechanism. IFITM3 expression was significantly overexpressed in the GC cell lines and GC tissues compared with corresponding normal controls by RT‑qPCR, western blot analysis and immunohistochemistry, and this overexpression was correlated with tumor differentiation, lymph node and distant metastasis, and advanced tumor node metastasis stages. Furthermore, knockdown of IFITM3 expression suppressed tumor cell migration, invasion and proliferation significantly in vitro, arrested tumor cells at the G0/G1 phase and reduced the cell numbers in the S phase of the cell cycle. We preliminarily confirmed that IFITM3 can be mediated by the activities of Wnt/β-catenin signaling. Further investigation revealed that silencing of IFITM3 effectively reversed the epithelial-to-mesenchymal transition (EMT) phenotype and reduced the activities of MMP-2 and MMP-9 expression. Taken together, these data suggested that IFITM3 is a potential therapeutic target for GC.

Influenza A virus-induced degradation of eukaryotic translation initiation factor 4B contributes to viral replication by suppressing IFITM3 protein expression.

Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein. Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent the host innate immune response, influenza virus has evolved multiple strategies for suppressing the production of IFNs. Here, we show that IAV infection induces lysosomal degradation of eIF4B protein; and eIF4B inhibits IAV replication by upregulating expression of interferon-induced transmembrane protein 3 (IFITM3), a key protein that protects the host from virus infection. Our finding illustrates a critical role of eIF4B in the host innate immune response and provides novel insights into the complex mechanisms by which influenza virus interacts with its host.