Interferon-induced Transmembrane Protein Research Articles

The Antiviral Activity of Interferon-Induced Transmembrane Proteins and Virus Evasion Strategies

The Antiviral Activity of Interferon-Induced Transmembrane Proteins and Virus Evasion Strategies

The Role and Mechanism of IFITM1 in Developing Acquired Cisplatin Resistance in Small Cell Lung Cancer

Platinum-based chemotherapies, historically the cornerstone of first-line treatment for small-cell lung cancer (SCLC), face a major hurdle: the frequent emergence of chemoresistance, notably to cisplatin (CDDP). Current understanding of the mechanisms driving CDDP resistance in SCLC is incomplete. Notably, Interferon inducible transmembrane protein1 (IFITM1) has been identified as a key player in the distant metastasis of SCLC. Analysis of The Cancer Genome Atlas (TCGA) database revealed that IFITM1 expression is markedly elevated in tumor tissues as compared to that from adjacent normal tissues, correlating with a worse prognosis for patients with SCLC. Our research focused on investigating the role of IFITM1 in the acquisition of cisplatin resistance in SCLC. Further clinical sample analysis highlighted a significant increase in IFITM1 levels in SCLC tissues from cisplatin-resistant patients versus those were responsive to CCDP treatment, with similar trends observed in cisplatin-resistant SCLC cells. Crucially, overexpression of IFITM1 reduced the sensitivity of SCLC cells to cisplatin, while silencing IFITM1 enhanced chemosensitivity in cisplatin-resistant strains. Our in vivo studies further confirmed that silencing IFITM1 significantly boosted the efficacy of cisplatin in inhibiting growth of subcutaneous tumors of NCI-H466/CDDP cells (cisplatin-resistant SCLC cells) in a mouse model. Mechanistically, IFITM1 appears to foster cisplatin resistance through activation of the Wnt/β-catenin pathway. In summary, our findings suggest that targeting IFITM1, alongside cisplatin treatment, could offer a promising therapeutic strategy to overcome resistance and improve outcomes for SCLC patients.

Interferon-Induced Transmembrane Protein-3 Rs12252-G Variant Increases COVID-19 Mortality Potential in Egyptian Population.

Coronavirus disease 2019 (COVID-19) represented an international health risk. Variants of the interferon-induced transmembrane protein-3 (IFITM3) gene can increase the risk of developing severe viral infections. This cross-sectional study investigated the association between IFITM3 rs12252A>G single nucleotide polymorphism (SNP) and COVID-19 severity and mortality in 100 Egyptian patients. All participants were subjected to serum interleukin-6 (IL-6) determination by ELISA and IFITM3 rs12252 genotyping by real-time polymerase chain reaction. Of all participants, 85.0% had the IFITM3 rs12252 homozygous AA genotype, whereas 15.0% had the heterozygous AG genotype. None of our participants had the homozygous GG genotype. The IFITM3 rs12252A allele was found in 92.5% and the G allele in only 7.5%. There was no significant association (p > 0.05) between the IFITM3 rs12252 SNP and COVID-19 severity, intensive care unit (ICU) admission, or IL-6 serum levels. The heterozygous AG genotype frequency showed a significant increase among participants who died (32.0%) compared with those who had been cured (9.3%). The mutant G allele was associated with patients' death. Its frequency among cured participants was 8.5%, whereas in those who died was 24.2% (p = 0.024) with 3.429 odds ratio [95% confidence interval: 1.1-10.4]. In conclusion, this study revealed a significant association between the G allele variant of IFITM3 rs12252 and COVID-19 mortality. However, results were unable to establish a significant link between rs12252 polymorphism, disease severity, ICU admission, or serum IL-6 levels.

Inflammatory pathways confer resistance to chemoradiotherapy in anal squamous cell carcinoma

Anal squamous cell carcinoma (ASCC) is associated with immunosuppression and infection with human papillomavirus (HPV). Response to standard chemoradiotherapy (CRT) varies considerably. A comprehensive molecular characterization of CRT resistance is lacking, and little is known about the interplay between tumor immune contexture, host immunity, and immunosuppressive and/or immune activating effects of CRT. Patients with localized ASCC, treated with CRT at three different sites of the German Cancer Consortium (DKTK) were included. Patient cohorts for molecular analysis included baseline formalin fixed paraffin embedded biopsies for immunohistochemistry (n = 130), baseline RNA sequencing (n = 98), peripheral blood immune profiling (n = 47), and serum cytokine measurement (n = 35). Gene set enrichment analysis showed that pathways for IFNγ, IFNα, inflammatory response, TNFα signaling via NF-κB, and EMT were significantly enriched in poor responders (all p < 0.001). Expression of interferon-induced transmembrane protein 1 (IFITM1), both on mRNA and protein levels, was associated with reduced Freedom from locoregional failure (FFLF, p = 0.037) and freedom from distant metastasis (FFDM, p = 0.014). An increase of PD-L1 expression on CD4+ T-cells (p < 0.001) and an increase in HLA-DR expression on T-cells (p < 0.001) was observed in the peripheral blood after CRT. Elevated levels of regulatory T-cells and CXCL2 were associated with reduced FFLF (p = 0.0044 and p = 0.004, respectively). Inflammatory pathways in tissue in line with elevated levels of regulatory T-cells and CXCL2 in peripheral blood are associated with resistance to CRT. To counteract this resistance mechanism, the RADIANCE randomized phase-2 trial currently tests the addition of the immune checkpoint inhibitor durvalumab to standard CRT in locally advanced ASCC.

Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

Cooperative interaction of interferon regulatory factor -1 and bromodomain-containing protein 4 on RNA polymerase activation for intrinsic innate immunity.

The human orthopneumovirus, Respiratory Syncytial Virus (RSV), is the causative agent of severe lower respiratory tract infections (LRTI) and exacerbations of chronic lung diseases. In immune competent hosts, RSV productively infects highly differentiated epithelial cells, where it elicits robust anti-viral, cytokine and remodeling programs. By contrast, basal cells are relatively resistant to RSV infection, in part, because of constitutive expression of an intrinsic innate immune response (IIR) consisting of a subgroup of interferon (IFN) responsive genes. The mechanisms controlling the intrinsic IIR are not known. Here, we use human small airway epithelial cell hSAECs as a multipotent airway stem cell model to examine regulatory control of an intrinsic IIR pathway. We find hSAECs express patterns of intrinsic IIRs, highly conserved with pluri- and multi-potent stem cells. We demonstrate a core intrinsic IIR network consisting of Bone Marrow Stromal Cell Antigen 2 (Bst2), Interferon Induced Transmembrane Protein 1 (IFITM1) and Toll-like receptor (TLR3) expression are directly under IRF1 control. Moreover, expression of this intrinsic core is rate-limited by ambient IRF1• phospho-Ser 2 CTD RNA Polymerase II (pSer2 Pol II) complexes binding to their proximal promoters. In response to RSV infection, the abundance of IRF1 and pSer2 Pol II binding is dramatically increased, with IRF1 complexing to the BRD4 chromatin remodeling complex (CRC). Using chromatin immunoprecipitation in IRF1 KD cells, we find that the binding of BRD4 is IRF1 independent. Using a small molecule inhibitor of the BRD4 acetyl lysine binding bromodomain (BRD4i), we further find that BRD4 bromodomain interactions are required for stable BRD4 promoter binding to the intrinsic IIR core promoters, as well as for RSV-inducible pSer2 Pol II recruitment. Surprisingly, BRD4i does not disrupt IRF1-BRD4 interactions, but disrupts both RSV-induced BRD4 and IRF1 interactions with pSer2 Pol II. We conclude that the IRF1 functions in two modes- in absence of infection, ambient IRF1 mediates constitutive expression of the intrinsic IIR, whereas in response to RSV infection, the BRD4 CRC independently activates pSer2 Pol II to mediates robust expression of the intrinsic IIR. These data provide insight into molecular control of anti-viral defenses of airway basal cells.

Abstract 6159: Single cell analysis of circulating peripheral blood mononuclear cells in African American men with prostate cancer and their association with lethal disease

Abstract Prostate cancer is a leading cause of cancer-related deaths among men in the United States and affects African American (AA) men more so than other men. We and others previously described a distinct tumor immunobiology in AA prostate cancer patients. Furthermore, we discovered a serum proteome-defined suppression of tumor immunity signature that is prevalent among these patients and associated with lethal prostate cancer. In the current study, we sought to investigate the composition of circulating peripheral blood mononuclear cells (PBMCs) in AA and European American (EA) prostate cancer patients and their association with patient group and lethal prostate cancer. We obtained high-quality RNA-sequencing data for 277336 single cells representing circulating PBMCs from 59 AA and EA men. In the analysis, being an AA patients associated with an increased abundance of exhausted T cells, higher numbers of plasma B cells and CD16+ monocytes. Gene signatures indicative of T cell exhaustion and Treg activity were generally elevated in AA PBMCs while T cell effector function features were decreased. Within the overall cell population, interferon gene signatures were robustly elevated in AA than EA patients. This gene signature pattern that we observed in AA patients also associated with lethal prostate cancer, namely the T cell exhaustion, Treg activity and elevated interferon signaling gene signatures, may contribute to immune dysfunction and immune injury; pointing to potential clinical implications. Lastly, the interferon-induced transmembrane protein (IFITM3), interferon alpha inducible protein (IFI6), cysteine rich protein-1 (CRIP1), and the RAC family small GTPase2 (RAC2) were notably upregulated in AA patient-derived immune cells. In summary, we show that circulating immune cell populations and the associated gene signatures differ between men of African and European descent. Those elevated in AA patients correlated with lethal prostate cancer. Citation Format: Md Shakir Uddin Ahmed, Tiffany Dorsey, Stefan Ambs. Single cell analysis of circulating peripheral blood mononuclear cells in African American men with prostate cancer and their association with lethal disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6159.

Genome-wide CRISPR activation screen identifies JADE3 as an antiviral activator of NF-kB–dependent IFITM3 expression

The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape we conducted a gain of function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including JADE3 a protein involved in directing the histone acetyltransferase HBO1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogues JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well characterized antiviral gene Interferon-Induced Transmembrane Protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB dependent IFITM3 expression to restrict influenza A virus infection.

The antiviral role of largemouth bass STING against iridovirus infection

Stimulator of interferon gene (STING) plays a crucial role in the innate immune response against viral and bacterial pathogens. However, its function in largemouth bass iridovirus (LMBV) infection remains uncertain. Here, a STING homolog (MsSTING) from largemouth bass (Micropterus salmoides) was cloned and characterized. MsSTING encoded a 407-amino-acid polypeptide, which shared 84.08% and 41.45% identity with golden perch (Perca flavescens) and human (Homo sapiens) homologs, respectively. MsSTING contained four transmembrane domains and a conserved C-terminal domain. The mRNA level of MsSTING was significantly increased in response to LMBV infection in vitro. Subcellular localization observation indicated that MsSTING encoded a cytoplasmic protein, which co-localized predominantly with endoplasmic reticulum (ER) and partially with mitochondria. Moreover, its accurate localization was dependent on the N-terminal transmembrane motif (TM) domains. MsSTING was able to activate interferon (IFN) response, evidenced by the activation of IFN1, IFN3 and ISRE promoters by its overexpression in vitro. Mutant analysis showed that both the N-terminal and C-terminal domain of MsSTING were essential for its activation on IFN response. In addition, overexpression of MsSTING inhibited the transcription and protein levels of viral core genes, indicating that MsSTING exerted antiviral action against LMBV. Consistently, the inhibitory effects were significantly attenuated when the N-terminal or C-terminal domains of MsSTING was deleted. Furthermore, MsSTING overexpression upregulated the transcriptions of interferon-related genes and pro-inflammatory factors, including TANK-binding kinase 1(TBK1), interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), interferon stimulated exonuclease gene 20 (ISG20), interferon-induced transmembrane protein 1(IFITM1), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6). Together, MsSTING exerted antiviral action upon LMBV infection through positive regulation the innate immune response.

IFITM3 inhibits severe fever with thrombocytopenia syndrome virus entry and interacts with viral Gc protein.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever disease with high fatality rate of 10%-20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad-spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti-SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti-SFTSV function by blocking Gn/Gc-mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3-Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3-Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs-mediated anti-SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.

Analysis of Chicken IFITM3 Gene Expression and Its Effect on Avian Reovirus Replication.

Interferon-inducible transmembrane protein 3 (IFITM3) is an antiviral factor that plays an important role in the host innate immune response against viruses. Previous studies have shown that IFITM3 is upregulated in various tissues and organs after avian reovirus (ARV) infection, which suggests that IFITM3 may be involved in the antiviral response after ARV infection. In this study, the chicken IFITM3 gene was cloned and analyzed bioinformatically. Then, the role of chicken IFITM3 in ARV infection was further explored. The results showed that the molecular weight of the chicken IFITM3 protein was approximately 13 kDa. This protein was found to be localized mainly in the cytoplasm, and its protein structure contained the CD225 domain. The homology analysis and phylogenetic tree analysis showed that the IFITM3 genes of different species exhibited great variation during genetic evolution, and chicken IFITM3 shared the highest homology with that of Anas platyrhynchos and displayed relatively low homology with those of birds such as Anser cygnoides and Serinus canaria. An analysis of the distribution of chicken IFITM3 in tissues and organs revealed that the IFITM3 gene was expressed at its highest level in the intestine and in large quantities in immune organs, such as the bursa of Fabricius, thymus and spleen. Further studies showed that the overexpression of IFITM3 in chicken embryo fibroblasts (DF-1) could inhibit the replication of ARV, whereas the inhibition of IFITM3 expression in DF-1 cells promoted ARV replication. In addition, chicken IFITM3 may exert negative feedback regulatory effects on the expression of TBK1, IFN-γ and IRF1 during ARV infection, and it is speculated that IFITM3 may participate in the innate immune response after ARV infection by negatively regulating the expression of TBK1, IFN-γ and IRF1. The results of this study further enrich the understanding of the role and function of chicken IFITM3 in ARV infection and provide a theoretical basis for an in-depth understanding of the antiviral mechanism of host resistance to ARV infection.

Role of Viral Envelope Proteins in Determining Susceptibility of Viruses to IFITM Proteins.

Interferon-induced transmembrane proteins (IFITMs) are a family of proteins which inhibit infections of various enveloped viruses. While their general mechanism of inhibition seems to be non-specific, involving the tightening of membrane structures to prevent fusion between the viral envelope and cell membrane, numerous studies have underscored the importance of viral envelope proteins in determining the susceptibility of viruses to IFITMs. Mutations in envelope proteins may lead to viral escape from direct interaction with IFITM proteins or result in indirect resistance by modifying the viral entry pathway, allowing the virus to modulate its exposure to IFITMs. In a broader context, the nature of viral envelope proteins and their interaction with IFITMs can play a crucial role in the context of adaptive immunity, leading to viral envelope proteins that are more susceptible to antibody neutralization. The precise mechanisms underlying these observations remain unclear, and further studies in this field could contribute to a better understanding of how IFITMs control viral infections.

Interferon-Induced Transmembrane Protein 3 (IFITM3) gene Polymorphisms in COVID-19 patients in Assiut University Hospitals

Interferon-Induced Transmembrane Protein 3 (IFITM3) gene Polymorphisms in COVID-19 patients in Assiut University Hospitals

CEACAM6 and IFITM3 as potential biomarkers for pelareorep in pancreatic adenocarcinoma (PDAC).

684 Background: Pelareorep (P) is a proprietary formulation of live, replication-competent, naturally occurring Reovirus Type 3 Dearing strain. We previously performed a phase II trial of P with carboplatin (C) and paclitaxel (Pxl) vs C and Pxl alone in metastatic PDAC (NCT01280058). Although P did not improve progression-free survival compared to C and Pxl alone, some exceptional durable responses were seen in the P arm. Low levels of carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) mRNA expression were associated with prolonged PFS in P-treated pts (p=0.05) and there was a 3.3 times higher fold change in Interferon-inducible transmembrane protein 3 (IFITM3) expression in the pts doing poorly versus responders (nominal p&lt;0.05) in P arm. Here we investigate mechanisms of resistance to P infection mediated through CEACAM6 and IFITM3 in a panel of pancreatic ductal adenocarcinoma cell lines. Methods: Prior studies noted CEACAM6 and IFITM3 may restrict cell entry of certain enveloped viruses. CEACAM6 and IFITM3 protein levels were measured in PDAC cell lines using Western blot. Flow cytometry was used to assess P infectivity. We have generated multiple knockdowns of CEACAM6 and IFITM3 in PDAC cell lines to determine changes in P infectivity and other inflammatory signalling pathways. Live cell imaging was used to assess cell viability. Results: Basal expression of CEACAM6 was measured in a panel of 4 PDAC cells (PANC10.5, HPAFII, BxPC3, CFPAC1). Panc10.05 had low basal expression nor could expression be induced by 24hr P infection. All other cell lines showed moderate to high basal expression that was inducible. P infectivity at 24hrs was significantly higher in Panc10.05 cells compared to high CEACAM6 expressing cell lines. There was varying basal expression of IFITM3, however all cell lines showed IFITM3 induction when exposed to P. Of the 3 cell lines that also expressed high levels of CEACAM6 (HPAFII, CFPAC1, BxPC3), low basal expression of IFITM3 was strongly associated with increased sensitivity to P infection and decreased viability at 72hrs, which could be induced by knockdown of IFITM3 in high expressing cell lines. Neither C or Pxl altered levels of CEACAM6 or IFITM3 in any of the four cell lines. Conclusions: High CEACAM6 and IFITM3 play a role in P viral infectivity across multiple pancreatic cell lines, confirming results obtained in translational datasets. Further analysis is ongoing to elucidate mechanism and identify pathways to alter expression of CEACAM6 and IFITM3 and enhance P infectivity in future trials.

Proteomic detection of COX-2 pathway-related factors in patients with adenomyosis.

Investigating the relationship between cyclooxygenase-2 (COX-2) pathway-related factors and clinical features in patients with adenomyosis by proteomics could provide potential therapeutic targets. This study recruited 40 patients undergoing surgical hysterectomy and pathological diagnosis of adenomyosis, collected ectopic endometrial specimens, and recorded clinical data. The expression levels of COX-2 in ectopic uterus lesions were detected using the immunohistochemical (IHC) SP method. The 40 samples were then divided into a COX-2 low or high expression group. Five samples with the most typical expression levels were selected from each of the two groups and the differential proteins between the two groups were identified using label-free quantitative proteomics. WW domain-binding protein 2 (WBP2), interferon induced transmembrane protein 3 (IFITM3), and secreted frizzled-related protein 4 (SFRP4) were selected for further verification, and their relationships with COX-2 and clinical characteristics were analyzed. There were statistically significant differences in the expression of WBP2, IFITM3, and SFRP4 between the COX-2 low and high expression groups (P < 0.01). The expressions of COX-2, IFITM3, and SFRP4 were significantly correlated with dysmenorrhea between the two groups (P < 0.05), but not with uterine size or menstrual volume (P > 0.05). However, there was no significant correlation between the expression of WBP2 and dysmenorrhea, uterine size, and menstruation volume in both the high expression and low expression groups (P > 0.05). COX-2, IFITM3, SFRP4, and WBP2 may be involved in the pathogenesis of adenomyosis. COX-2, IFITM3, and SFRP4 may serve as potential molecular biomarkers or therapeutic targets in dysmenorrhea in patients with early adenomyosis.

IFITM3 promotes glioblastoma stem cell-mediated angiogenesis via regulating JAK/STAT3/bFGF signaling pathway

Interferon-induced transmembrane protein 3 (IFITM3) has been previously verified to be an endosomal protein that prevents viral infection. Recent findings suggested IFITM3 as a key factor in tumor invasion and progression. To clarify the role and molecular mechanism of IFITM3 in Glioblastoma multiforme (GBM) progression, we investigated the expression of IFITM3 in glioma datasets culled from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA). Primary GBM stem cells (GSCs) were cultured and identified in vitro. Loss-of-function and gain-of-function experiments were established by using shRNAs and lentiviral vectors targeting IFITM3. Co-culture system of GSCs and vascular endothelial cells was constructed in a Transwell chamber. Tube formation and spheroid-based angiogenesis assays were performed to determine the angiogenic capacity of endothelial cells. Results revealed that IFITM3 is elevated in GBM samples and predictive of adverse outcome. Mechanistically, GSCs-derived IFITM3 causes activation of Jak2/STAT3 signaling and leads to robust secretion of bFGF into tumor environment, which eventually results in enhanced angiogenesis. Taken together, these evidence indicated IFITM3 as an essential factor in GBM angiogenesis. Our findings provide a new insight into mechanism by which IFITM3 modulates GBM angiogenesis.

Evaluation of long non-coding RNAs EGOT, NRAV, NRIR and mRNAs ISG15 and IFITM3 expressions in COVID-19 patients

Individuals with Coronavirus Disease 2019 (COVID-19) may show no symptoms to moderate or severe complications. This variation may be due to differences in the strength of the immune response, including a delayed interferon (IFN) response in asymptomatic patients and higher IFN levels in severe patients. Some long non-coding RNAs (lncRNAs), as regulators of the IFN pathway, may contribute to the emergence of different COVID-19 symptoms. This study aimed to comparatively investigate the relationship between lncRNAs (eosinophil granule ontogeny transcript (EGOT), negative regulator of antiviral response (NRAV), and negative regulator of interferon response (NRIR)), alongside interferon-stimulated genes (ISGs) like ISG-15 and interferon-induced transmembrane protein 3 (IFITM3) in COVID-19 patients with asymptomatic, moderate, and severe symptoms. Buffy coat samples were collected from 17 asymptomatic, 23 moderate, 22 severe patients, and 44 healthy controls. Quantitative real-time PCR was utilized to determine the expression levels. In a comparison between COVID-19 patients and healthy individuals, higher expression levels of EGOT and NRAV were observed in severe and moderate patients. NRIR expression was increased across all patient groups. Meanwhile, ISG15 expression decreased in all patient groups, and the moderate group showed a significant decrease in IFITM3 expression. Comparing COVID-19 patient groups, EGOT expression was significantly higher in moderate COVID-19 patients compared to asymptomatic patients. NRAV was higher in moderate and severe patients compared to asymptomatic. NRIR levels did not differ significantly between the COVID-19 patient groups. ISG15 was higher in moderate and severe patients compared to asymptomatic. IFITM3 expression was significantly higher in severe patients compared to the moderate group. In severe COVID-19 patients, EGOT expression was positively correlated with NRAV levels. EGOT and NRAV showed a significant positive correlation in asymptomatic patients, and both were positively correlated with IFITM3 expression. This study suggests that EGOT, NRAV, NRIR, ISG15, and IFITM3 may serve as diagnostic biomarkers for COVID-19. The lncRNA NRAV may be a good biomarker in a prognostic panel between asymptomatic and severe patients in combination with other high-sensitivity biomarkers. EGOT, NRAV, and ISG15 could also be considered as specific biomarkers in a prognostic panel comparing asymptomatic and moderate patients with other high-sensitivity biomarkers.

Proteomic profiling of ovarian clear cell carcinomas identifies prognostic biomarkers for chemotherapy.

Clear cell ovarian carcinoma (CCOC) is a relatively rare subtype of ovarian cancer (OC) with high degree of resistance to standard chemotherapy. Little is known about the underlying molecular mechanisms, and it remains a challenge to predict its prognosis after chemotherapy. Here, we first analyzed the proteome of 35 formalin-fixed paraffin-embedded (FFPE) CCOC tissue specimens from a cohort of 32 patients with CCOC (H1 cohort) and characterized 8697 proteins using data-independent acquisition mass spectrometry (DIA-MS). We then performed proteomic analysis of 28 fresh frozen (FF) CCOC tissue specimens from an independent cohort of 24 patients with CCOC (H2 cohort), leading to the identification of 9409 proteins with DIA-MS. After bioinformatics analysis, we narrowed our focus to 15 proteins significantly correlated with the recurrence free survival (RFS) in both cohorts. These proteins are mainly involved in DNA damage response, extracellular matrix (ECM), and mitochondrial metabolism. Parallel reaction monitoring (PRM)-MS was adopted to validate the prognostic potential of the 15 proteins in the H1 cohort and an independent confirmation cohort (H3 cohort). Interferon-inducible transmembrane protein 1 (IFITM1) was observed as a robust prognostic marker for CCOC in both PRM data and immunohistochemistry (IHC) data. Taken together, this study presents a CCOC proteomic data resource and a single promising protein, IFITM1, which could potentially predict the recurrence and survival of CCOC.

A Knockout of the IFITM3 Gene Increases the Sensitivity of WI-38 VA13 Cells to the Influenza A Virus.

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 gene, whose product limits the entry of a virus into the cell by blocking the fusion of the viral and endosomal membranes. In this study, by means of genome-editing system CRISPR/Cas9, monoclonal cell lines with an IFITM3 knockout were obtained based on WI-38 VA13 cells (human origin). It was found that such cell lines are more sensitive to infection by influenza A viruses of various subtypes. Nevertheless, this feature is not accompanied by an increased titer of newly formed viral particles in a culture medium.

IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.