Bone Marrow Stromal Antigen 2 Research Articles

Neocarzilin Inhibits Cancer Cell Proliferation via BST-2 Degradation, Resulting in Lipid Raft-Trapped EGFR.

Neocarzilin (NCA) is a natural product exhibiting potent antimigratory as well as antiproliferative effects. While vesicle amine transport protein 1 (VAT-1) was previously shown to inhibit migration upon NCA binding, the molecular mechanisms responsible for impaired proliferation remained elusive. We here introduce a chemical probe closely resembling the structural and stereochemical features of NCA and unravel bone marrow stromal antigen 2 (BST-2) as one of the targets responsible for the antiproliferative effect of NCA in cancer cells. The antiproliferative mechanism of NCA was confirmed in corresponding BST-2 knockout (KO) HeLa cells, which were less sensitive to compound treatment. Vice versa, reconstitution of BST-2 in the KO cells again reduced proliferation upon NCA addition, comparable to that of wild-type (wt) HeLa cells. Whole proteome mass spectrometric (MS) analysis of NCA-treated wt and KO cancer cells revealed regulated pathways and showed reduced levels of BST-2 upon NCA treatment. In-depth analysis of BST-2 levels in response to proteasome and lysosome inhibitors unraveled a lysosomal degradation path upon NCA treatment. As BST-2 mediates the release of epidermal growth factor receptor (EGFR) from lipid rafts to turn on proliferation signaling pathways, reduced BST-2 levels led to attenuated phosphorylation of STAT3. Furthermore, fluorescence microscopy confirmed increased colocalization of EGFR and lipid rafts in the presence of NCA. Overall, NCA represents a versatile anticancer natural product with a unique dual mode of action and unconventional inhibition of proliferation via BST-2 degradation.

Abstract PO5-24-02: Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer

Abstract Background: A subpopulation of tumor cells known to have intrinsic resistance to therapies and contribute to metastasis function as “cancer stem”, or tumor-initiating, cells (TICs). TICs can be identified by various biomarkers, including fluorescent reporters for signaling pathways that regulate TIC function such as STAT signaling. However, standard TIC reporters have limitations. Their expression is unstable and there is no established method to follow TICs as they undergo cell state changes. Furthermore, we do not completely understand the transcriptional features of TICs. Improved methods of identifying TICs as well as the development of high-resolution transcriptional signatures are essential to understand TIC biology. An improved understanding of TIC biology can inform the development of novel therapeutics to selectively ablate the TIC compartment, thus improving response to standard cancer therapies. Methods: We screened a panel of PDX models to determine whether a STAT reporter identifies TICs in patient-derived xenograft (PDX) models. To augment existing STAT TIC reporters, we developed a two-component STAT signaling-specific lineage-tracing (LT) system. The first component labels active STAT signaling cells by expression of enhanced green fluorescent protein (EGFP+), followed by a self-cleaving P2A peptide and a TAM-inducible Cre-recombinase (4M67-EGFP-P2A-CreERT2). The second component is a constitutively expressed dual-color switching Cre-dependent reporter vector (EFS-loxPdsRedloxP-mNeptune2). We determined which LT cell populations (EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP−/mNeptune2+, and EGFP−/dsRed+) from SUM159 xenografts functioned as TICs. To probe TIC transcriptional features, we performed scRNA seq on the two tumor models with STAT TICs and a PDX model that did not have STAT TICs. We performed lineage trajectory analysis and differential gene expression analysis to characterize candidate TIC transcriptional signatures. To confirm whether these transcriptional features are associated with TICs, we identified a biomarker of this cell state (bone marrow stromal antigen 2 [BST2]) and explored the relationship between this biomarker and TICs. Results: We identified four PDX models with STAT reporter activity, one of which had STAT TICs. We validated our LT system in several models both in vitro and in vivo, then demonstrated EGFP+/mNeptune2+ from SUM159 xenografts are enriched for TICs and that lineage-tagged cells (mNeptune2+) function as proliferative early progenitor cells. scRNA seq of three xenograft models uncovered a distinct antiviral cell state in all three models that represent a more highly enriched TIC subpopulation. Critically, transcriptional features were shared between the antiviral cell states across xenograft models. To determine whether cells in this antiviral state represent TICs, we FACS-enriched BST2 cells (an IFN-stimulated cell surface protein that was a marker of the antiviral cell state) and demonstrated enrichment of the TIC population by mammosphere formation assays and limiting dilution transplantation. We demonstrated that BST2 expression is increased in residual PDX tumors following chemotherapy. Furthermore, we demonstrated similar antiviral cell states are present in human breast cancer scRNA seq datasets. Conclusions: These data demonstrate TICs adopt an antiviral cell state in some triple-negative breast cancers, and BST2 is a function marker of TICs in this cell state. Therefore, targeting genes associated with this cell state or associated antiviral pathways may represent a therapeutic vulnerability to eliminate TICs in some breast cancers. Citation Format: Eric Souto, Ping Gong, John Landua, Ram Srinivasan, Lacey Dobrolecki, Abhinaya Ganesan, Michael Lewis. Interferon-induced bone marrow stromal antigen 2 (BST2) is a functional tumor-initiating cell marker in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-02.

ATG5 selectively engages virus-tethered BST2/tetherin in an LC3C-associated pathway

Bone marrow stromal antigen 2 (BST2)/tetherin is a restriction factor thatreduces HIV-1 dissemination by tethering virus at the cell surface. BST2 also acts as a sensor of HIV-1 budding, establishing a cellular antiviral state. The HIV-1 Vpu protein antagonizes BST2 antiviral functions via multiple mechanisms, including the subversion of an LC3C-associated pathway, a key cell intrinsic antimicrobial mechanism. Here, we describe the first step of this viral-induced LC3C-associated process. This process is initiated at the plasma membrane through the recognition and internalization of virus-tethered BST2 by ATG5, an autophagy protein. ATG5 and BST2 assemble as a complex, independently of the viral protein Vpu and ahead of the recruitment of the ATG protein LC3C. The conjugation of ATG5 with ATG12 is dispensable for this interaction. ATG5 recognizes cysteine-linked homodimerized BST2 and specifically engages phosphorylated BST2 tethering viruses at the plasma membrane, in an LC3C-associated pathway. We also found that this LC3C-associated pathway is used by Vpu to attenuate the inflammatory responses mediated by virion retention. Overall, we highlight that by targeting BST2 tethering viruses, ATG5 acts as a signaling scaffold to trigger an LC3C-associated pathway induced by HIV-1 infection.

Identification of BST2 as a biomarker for alopecia areata in both mice and humans

Abstract Alopecia areata is an autoimmune disease characterized by aberrant non-scarring hair loss. 147 million people worldwide are currently diagnosed with alopecia areata with a lifetime incidence of 2.1%. The hair follicle is a site of immune privilege however in alopecia areata, CD4+ helper T cells and CD8+ cytotoxic T cells infiltrate and attack the hair follicle. To better understand the initiating mechanisms surrounding alopecia areata, we examined the function of epidermal γδ T cells and dermal macrophages in the initiation of or protection from the disease. We show that epidermal γδ T cells and keratinocytes are elevated in the skin of C3H/HeJ mice with alopecia areata. Analysis of publicly available single cell RNA sequencing data delineated the top differentially expressed genes of epidermal γδ T cells and dermal macrophages. Bone marrow stromal antigen 2 (BST2) is significantly upregulated among both cell types and is a novel gene associated with alopecia areata. Using immunofluorescence microscopy we determined that a small population of hair follicle-associated epidermal γδ T cells express BST2 in C3H/HeJ mice and C57Bl/6J mice without alopecia areata. We have also identified a significant increase in BST2 expression in skin sections from mice affected by alopecia areata. BST2+CD11b+ macrophages upregulate genes associated with antiviral activity indicating an association between the pathogenesis of alopecia areata and infectious viral particles. BST2+ epidermal γδ T cells upregulate genes associated with FAK signaling in alopecia areata, which indicates a role for FAK in the increased number of epidermal T cells. These studies identify a role for BST2 in alopecia areata that is mediated by epidermal γδ T cells and macrophages.

BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway.

Oral squamous cell carcinoma (OSCC) is one of the main types of head and neck squamous cell carcinoma. Although progress has been made in treating OSCC, it remains a threat to human health, and novel therapeutic strategies are needed to extend the lifespan of patients with OSCC. The present study, evaluated whether bone marrow stromal antigen2 (BST2) and STAT1 were potential therapeutic targets in OSCC. Small interfering RNA (siRNA) or overexpression plasmids were used to regulate BST2 or STAT1 expression. Western blotting and reverse transcription‑quantitative PCR were performed to assess changes in the protein and mRNA expression levels of signaling pathway components. The effects of BST2 and STAT1 expression changes on the migration, invasion and proliferation of OSCC cells were assessed using the scratch test assay, Transwell assay and colony formation assay invitro, respectively. Cell‑derived xenograft models were used to evaluate the impact of BST2 and STAT1 on the occurrence and development of OSCC invivo. Finally, it was demonstrated that BST2 expression was significantly upregulated in OSCC. Furthermore, it was demonstrated that high expression of BST2 in OSCC contributed to the metastasis, invasion and proliferation of OSCC cells. Moreover, it was demonstrated that the promoter region of BST2 was regulated by the transcription factor STAT1, and that the STAT1/BST2 axis could affect the behavior of OSCC via the AKT/ERK1/2 signaling pathway. Invivo studies also demonstrated that STAT1 downregulation inhibited OSCC growth by down‑regulating BST2 expression via the AKT/ERK1/2 signaling pathway.

The roles of BST-2 in murine B cell development and on virus propagation.

The bone marrow (BM) stromal cell antigen-2 (BST-2), also known as tetherin, CD317, PDCA-1, or HM1.24, is a membrane protein overexpressed in several types of tumors and may act as a promising target for cancer treatment via antibody-dependent cellular cytotoxicity. BST-2 is also expressed in human BM stromal cells (BMSC), which support B cell development. While the activity of BST-2 as an antiviral factor has been demonstrated, the expression patterns and the role of BST-2 on B-cell development and activation have not been investigated, especially in vivo. In this study, Bst2 knockout (Bst2-/- ) mice were generated to assess the role of BST-2 on B cell development and activation. It was observed that BST-2 was not expressed in BMSC or all B cell progenitors even in wild-type mice and does not play a significant role in B cell development. In addition, the loss of BST-2 had no effect on B cell activation. Furthermore and in contrast to the well-known antiviral role of BST-2, infection of vesicular stomatitis Indiana virus to the BM cells collected from the Bst2-/- mice produced less infectious virus compared with that from the WT mice. These results suggest that murine BST-2 is different from human BST-2 in the expression pattern, physiological function, in vivo, and might possess positive role on VSV replication.

Increased proliferation of epidermal gamma delta T cells and expression of the transmembrane protein, BST2, in Alopecia areata

Abstract Alopecia areata is an autoimmune disease characterized by patchy non-scarring hair loss in affected individuals. Typically, research has focused on hair follicle infiltration and disruption via CD8+ and CD4+ T cells. Our research attempts to elucidate the role of resident epidermal γδ T cells in disease. Analysis of publicly available bioinformatics data shows a statistically significant downregulation in a number of immune-related genes after patients were treated with a JAK inhibitor, Tofacitinib. We have focused on one of these proteins, bone marrow stromal antigen 2 (BST2), in vitro and in vivo using the C3H mouse model of alopecia areata. We have identified populations of epidermal γδ T cells expressing BST2 in the follicle using immunofluorescence microscopy. In addition, BST2 expression is upregulated in unaffected skin from mice with active alopecia areata suggesting BST2 expression may precede loss of hair follicle immune privilege. Furthermore, epidermal γδ T cell number is increased in skin affected by alopecia areata. Together our findings suggest that BST2 is expressed by epidermal T cells prior to hair loss and is followed by an increase in epidermal γδ T cell number. Our lab is focused on how these changes in epidermal γδ T cell number and BST2 expression impacts disease. Supported by grants from NIH Supported by grants from NSF

Bone marrow stromal antigen 2 (BST-2) genetic variants influence expression levels and disease outcome in HIV-1 chronically infected patients

BackgroundBone marrow stromal antigen 2 (BST-2) also known as Tetherin (CD317/HM1.24), is a host restriction factor that blocks the release of HIV-1 virions from infected cells. Previous studies reported that BST-2 genetic variants or single nucleotide polymorphims (SNPs) have a preventative role during HIV-1 infection. However, the influence of BST-2 SNPs on expression levels remains unknown. In this study, we investigated the influence of BST-2 SNPs on expression levels and disease outcome in HIV-1 subtype C chronically infected antiretroviral therapy naïve individuals.ResultsWe quantified BST-2 mRNA levels in peripheral blood mononuclear cells (PBMCs), determined BST-2 protein expression on the surface of CD4+ T cells using flow cytometry and genotyped two intronic single nucleotide polymorphisms (SNPs) rs919267 and rs919266 together with one SNP rs9576 located in the 3’ untranslated region (UTR) of bst-2 gene using TaqMan assays from HIV-1 uninfected and infected participants. Subsequently, we determined the ability of plasma antibody levels to mediate antibody-dependent cellular phagocytosis (ADCP) using gp120 consensus C and p24 subtype B/C protein. Fc receptor-mediated NK cell degranulation was evaluated as a surrogate for ADCC activity using plasma from HIV-1 positive participants. BST-2 mRNA expression levels in PBMCs and protein levels on CD4+ T cells were lower in HIV-1 infected compared to uninfected participants (p = 0.075 and p < 0.001, respectively). rs919267CT (p = 0.042) and rs919267TT (p = 0.045) were associated with lower BST-2 mRNA expression levels compared to rs919267CC in HIV-1 uninfected participants. In HIV-1 infected participants, rs919267CT associated with lower CD4 counts, (p = 0.003), gp120-IgG1 (p = 0.040), gp120-IgG3 (p = 0.016) levels but higher viral loads (p = 0.001) while rs919267TT was associated with lower BST-2 mRNA levels (p = 0.046), CD4 counts (p = 0.001), gp120-IgG1 levels (p = 0.033) but higher plasma viral loads (p = 0.007). Conversely, rs9576CA was associated with higher BST-2 mRNA expression levels (p = 0.027), CD4 counts (p = 0.079), gp120-IgG1 (p = 0.009), gp120-IgG3 (p = 0.039) levels but with lower viral loads (p = 0.037).ConclusionOur findings show that bst-2 SNPs mediate BST-2 expression and disease outcome, correlate with gp120-IgG1, gp120-IgG3 levels but not p24-IgG levels, ADCC and ADCP activity.Graphical

Bone Marrow Stromal Antigen 2 Facilitates Activation of Hematopoietic Stem Cell through the Induction of ERK Signaling

Chronic infections are prevalent worldwide and contribute to bone marrow failure syndromes. Long-term homeostatic production of all blood cells is dependent on the ability of hematopoietic stem cells (HSCs) to remain mostly quiescent and, upon activation, must carefully balance between their ability to self-renew and differentiate. However, infections trigger inflammatory cytokines, such as interferon-gamma (IFNy), that facilitate HSC proliferative exhaustion by disrupting both quiescence and self-renewal, thereby contributing to cytopenias and bone marrow failure syndromes. Our previously published results indicate that the IFN-induced surface protein, Bone Marrow Stromal Antigen 2 (BST2), facilitates HSC activation, and the loss of BST2 protected against the depletion of HSCs during chronic infection. However, the mechanism by which BST2 impacts HSCs activation is unknown. BST2 is known to promote activation of intracellular signaling in both dendritic cells and cancer cell types. In particular BST2 is a known activator of the NFkB signaling pathway. We hypothesized that BST2 could promote activation of HSCs through the induction of intracellular signaling pathways.Intracellular signaling pathways have been shown to be important for the activation of not only HSCs but also downstream immune cells. Indeed, the loss of BST2 has been shown to impair the function of T cells, NK cells, and antigen presenting cells in the presence of viral infections via impaired induction of NFkB signaling. However, there have been no studies on the role of BST2 during bacterial infections. To evaluate the role of BST2 in hematopoiesis during a bacterial infection, we obtained BST2KO mice and characterized hematopoietic populations in the presence or absence of Mycobacterium avium infection, a model bacterial infection that generates high levels of IFNy. We observed no changes in activation or production of cytokines from macrophage, NK, CD8, or neutrophil populations. Surprisingly we observed enhanced dendritic cell activation at day 15, 30, and 42 as measured by co-stimulatory molecules and MHC II expression. Additionally, we observed increased tetramer-specific T cell numbers early on during the infection. Interestingly, these results pair with decreased bacterial load at day 30 and day 42 despite having the similar spleen sizes. These results suggest that BST2 regulates the activation of dendritic cells, which may trigger more robust earlier T cell responses early in the course of infection.Our previous work showed that the loss of BST2 protects against HSC activation by displacement of HSCs from CXCL12-abundant reticular (CAR) cells in the niche. To determine whether HSC activation is related to distancing from the vascular niche or increased proximity to an activated osteoblastic niche, we performed intravital imaging using osteocalcin-GFP reporter mice. We observed no change in proximity to the osteoblastic niche after administration of recombinant IFNy using either WT or BST2KO HSCs. These results suggest that the effects of BST2 may be more related to distancing from CAR cells than migration towards other niche locations.To evaluate the mechanisms underlying BST2-dependent HSC activation, we performed RNA sequencing on WT versus BST2KO HSCs in the presence of IFNy. GSEA-pathway analysis of differentially expressed genes revealed that BST2KO HSCs have a decreased response to IFNy and diminished activation of the P13K-AKT-MTOR signaling pathways. Western blot analysis on hematopoietic stem cell progenitor cells (HSPCs) indicate a decrease in phosphorylation of the ERK signaling pathway in BST2KO HSPCs. These results were analogous to work performed in 32D cells in vitro, in which ERK-phosphorylation has been shown to be important for both HSC maintenance and proliferation. Future studies are aimed at assessing the impact of ERK inhibition on HSC activation during various inflammatory stresses.Collectively our results show that the loss of BST2 in downstream immune cells surprisingly enhances dendritic cell and T cell activation in response to bacterial infection. BST2 enhances HSC activation through activation of downstream ERK signaling. Future targeting of BST2 and/or ERK signaling could have important consequences for patients that suffer from cytopenias, bone marrow failure, and engraftment failure due to high levels of inflammatory stimuli. DisclosuresNo relevant conflicts of interest to declare.

Total ginsenosides induce autophagic cell death in cervical cancer cells accompanied by downregulation of bone marrow stromal antigen-2.

Ginsenosides are important active components in Panax ginseng. In the present study, total ginsenosides (TGNs) were demonstrated to enhance autophagy by promoting acidic vacuole organelle formation, recruitment of enhanced green fluorescent protein-microtubule-associated protein light chain 3 and expression of autophagy-related factors in cervical cancer cell lines. TGN markedly increased the expression of p62 at the transcriptional level, but decreased p62 protein expression in the presence of actinomycin D. The autophagic regulatory effect was reversible. TGN (≤120 µg/ml) did not affect the proliferation of cervical cancer cells under normal culture conditions, but markedly inhibited the growth of serum-deprived cells. Treatment with an inhibitor of autophagy (3-methyladenine) impaired TGN-induced cell death. This suggested that TGN caused autophagic cell death. In addition, western blot analysis demonstrated that the protein level of bone marrow stromal antigen-2 (BST-2) was downregulated by TGN. Upregulation of BST-2 reduced cell death. The results of the combined actions of various monomeric ginsenosides in TGN provide the molecular basis to develop TGN as a promising candidate for cancer therapy.

Bone Marrow Stromal Antigen 2 Is Critical for IFNy-Dependent Hematopoietic Stem Cell Activation

Bone marrow failure is a significant complication of many chronic infections, which affect a third of the world's population. The inflammatory cytokine interferon-gamma (IFNy) contributes to bone marrow failure syndromes by activating hematopoietic stem cells (HSCs) and impairing their self-renewal. IFNy upregulation during many chronic infections such as tuberculosis, HIV and hepatitis B directly depletes hematopoietic stem cells (HSCs). The mechanisms by which IFNy drives the loss of quiescence and ultimate exhaustion of HSCs remain poorly understood, but may be related to changes in the interaction between HSCs and the bone marrow (BM) microenvironment, or BM niche. Current evidence suggests that quiescent HSCs reside predominantly in the vascular niche, where the production of stem cell factor (SCF) from endothelial cells and CXCL12 from perivascular CXCL12 abundant reticular (CAR) stromal cells are critical for maintaining their quiescence. The goal of our work was to determine whether IFNy signaling alters HSC interactions within the niche. We performed transcriptomic analysis of IFNy-stimulated HSCs and focused on changes in cell-surface expressed genes that may influence HSC-niche interactions. Our analysis revealed Bone Marrow Stromal Antigen 2 (BST2) as the only surface protein upregulated on HSCs upon 24-hour IFNy stimulation. To study the effects of BST2 on HSC-niche interactions, we performed intravital imaging using two reporter mouse models: CXCL12-GFP reporter mice in which CAR cells are labeled with GFP, and CXCL12-GFP Krt18-Cre LSL-tdTomato dual reporter mice in which CAR cells are labelled with GFP and HSCs are labeled with tdTomato. After exogenously labeling and transferring HSCs into CXCL12-GFP mice or endogenously labeling HSCs in the CXCL12-GFP Krt18-Cre LSL-tdTomato mice, we observed that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. These findings are consistent with other reports that chemotherapeutic and inflammatory stress disrupts HSC interactions with the niche and promotes HSC migration. Conversely, we observed no change in HSC distancing from CAR cells after IFNy stimulation of IFNy-receptor deficient HSCs, suggesting that the observed HSC displacement was due to a cell autonomous mechanism. These changes were not due to a loss of CXCL12 receptor (CXCR4) expression or disrupted capacity of HSCs to migrate towards CXCL12. Interestingly, Intravital imaging using BST2-deficient HSCs revealed that BST2 KO HSCs do not re-localize from CAR cells during IFNy stimulation. Increased BST2 expression has been linked to the migration, adhesion and metastasis of various cancer cells and we explored whether it could serve a similar role in protein binding in HSCs. Using in vitro plate binding assays, we found that IFNy-treatment promoted increased HSC binding to E-selectin via BST2, as well as increased HSC homing to the bone marrow, a property that is dependent on E-selectin binding. Finally, to determine whether BST2 affects IFNy-dependent HSC activation we performed cell cycle analysis of WT and BST2 KO HSCs. We discovered that the loss of BST2 protects against HSC activation during Mycobacterium avium infection. Furthermore, HSC depletion during chronic infection was mitigated in BST2 KO mice. Our data identifies BST2 as a key protein that influences niche relocalization and activation in response to inflammatory stimulation. This study expands our understanding of factors that contribute to HSC activation and loss of quiescence. These findings could shed light on novel therapeutic interventions for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

Bone Marrow Stromal Antigen 2 is a Potential Unfavorable Prognostic Factor for High-Grade Glioma.

BackgroundBone marrow stromal antigen 2 (BST2) is considered as a transmembrane glycoprotein and plays essential roles in innate immunity. It has been recently reported that up-regulation of BST2 was associated with the development of breast carcinoma. However, the clinical significance of BST2 in glioma has not been identified. The purpose of the present study is to explore the expression pattern and the role of BST2 in the progression of high-grade glioma.MethodsExpression levels of BST2 were tested in glioma tissues by analyzing the GEO database and immunohistochemistry staining. The prognostic role of BST2 in glioma was evaluated through univariate and multivariate analyses. In vitro and in vivo assays were conducted to confirm the role of BST2 on promoting glioma proliferation.ResultsThe mRNA level of BST2 was higher in glioma tissues than that in nontumorous brain tissues. High protein level of BST2 was correlated with larger tumor size and advanced WHO grade. Glioma patients with a high BST2 level had worse overall survival. In addition, BST2 was defined as an independent risk factor for glioma prognosis. Cellular and xenograft studies revealed that BST2 can significantly promote glioma proliferation.ConclusionOur study revealed that a high BST2 expression level was closely related to the unfavorable clinical features and poor prognosis of high-grade glioma patients. BST2 may serve as an invaluable prognostic indicator and novel therapeutic target for glioma treatment considering its membrane localization.

BST2 Promotes Tumor Growth via Multiple Pathways in Hepatocellular Carcinoma

Bone marrow stromal antigen 2 (BST2) is a transmembrane glycoprotein and plays an essential role in innate immunity. Here we firstly found that BST2 expression was significantly elevated in hepatocellular carcinoma (HCC) tissues. High BST2 was closely related to the larger tumor size and more tumor number. Moreover, HCC patients with higher expression of BST2 had poorer overall survival and BST2 was identified as an independent unfavorable prognosis factor. Finally, we demonstrated that BST2 can promote proliferation capacity of tumor cells. In conclusion, HCC patients with higher BST2 expression were more predisposed to poorer clinical symptoms and unfavorable prognosis.

Lymphocyte‐specific protein 1 (LSP1) regulates bone marrow stromal cell antigen 2 (BST‐2)‐mediated intracellular trafficking of HIV‐1 in dendritic cells

Human immunodeficiency virus type 1 (HIV-1) subverts intracellular trafficking pathways to avoid its degradation and elimination, thereby enhancing its survival and spread. The molecular mechanisms involved in intracellular transport of HIV-1 are not yet fully defined. We demonstrate that the actin-binding protein lymphocyte-specific protein 1 (LSP1) interacts with the interferon-inducible protein bone marrow stromal antigen 2 (BST-2) in dendritic cells (DCs) to facilitate both endocytosis of surface-bound HIV-1 and the formation of early endosomes. Analysis of the molecular interaction between LSP1 and BST-2 reveals that the N terminus of LSP1 interacts with BST-2. Overall, we identify a novel mechanism of intracellular trafficking of HIV-1 in DCs centering on the LSP1/BST-2 complex. We also show that the HIV-1 accessory protein Vpu subverts this pathway by inducing proteasomal degradation of LSP1, augmenting cell-cell transmission of HIV-1.

Human BST-2/tetherin inhibits Junin virus release from host cells and its inhibition is partially counteracted by viral nucleoprotein.

Bone marrow stromal cell antigen-2 (BST-2), also known as tetherin, is an interferon-inducible membrane-associated protein. It effectively targets enveloped viruses at the release step of progeny viruses from host cells, thereby restricting the further spread of viral infection. Junin virus (JUNV) is a member of Arenaviridae, which causes Argentine haemorrhagic fever that is associated with a high rate of mortality. In this study, we examined the effect of human BST-2 on the replication and propagation of JUNV. The production of JUNV Z-mediated virus-like particles (VLPs) was significantly inhibited by over-expression of BST-2. Electron microscopy analysis revealed that BST-2 functions by forming a physical link that directly retains VLPs on the cell surface. Infection using JUNV showed that infectious JUNV production was moderately inhibited by endogenous or exogenous BST-2. We also observed that JUNV infection triggers an intense interferon response, causing an upregulation of BST-2, in infected cells. However, the expression of cell surface BST-2 was reduced upon infection. Furthermore, the expression of JUNV nucleoprotein (NP) partially recovered VLP production from BST-2 restriction, suggesting that the NP functions as an antagonist against antiviral effect of BST-2. We further showed that JUNV NP also rescued the production of Ebola virus VP40-mediated VLP from BST-2 restriction as a broad spectrum BST-2 antagonist. To our knowledge, this is the first report showing that an arenavirus protein counteracts the antiviral function of BST-2.

Cancerous Phenotypes Exhibited by NIH/3T3 Fibroblasts Overexpressing BST‐2

Oncogenesis is a multistep process initiated by the accumulation of genetic and/or epigenetic changes that culminate in the transformation of normal cells into cancer cells. The genetic and/or epigenetic changes result in dysregulated gene expression signature that may involve key cancer driving genes such as Bone Marrow Stromal Antigen 2 (BST‐2). In this study, we sought to test the hypothesis that BST‐2, a type II transmembrane protein regulates early events of oncogenesis. BST‐2 is a multifaceted protein with both antiviral and proviral functions and has been implicated in promoting aggressive cancer cell behavior such as migration and invasion. Meta‐analysis of breast cancer datasets from The Cancer Genome Atlas (TCGA) show a statistically significant correlation between elevated BST‐2 expression and poor survival. In addition to its role in breast cancer progression, elevated BST‐2 expression correlates with poor prognosis in endometrial, brain, lung, and oral cavity cancers, further implicating elevated BST‐2 expression in cancer progression. However, the role of BST‐2 in regulating early oncogenic events is not well understood. Our approach consisted of stably overexpressing exogenous human BST‐2 in NIH/3T3 mouse embryonic fibroblasts that express very low endogenous mouse BST‐2 to generate 3T3BST‐2 cells. The control cells were stably transfected with pCDNA3.1 to generate 3T3pCDNA cells. The cells were tested for phenotypes associated with early oncogenic events under two‐dimensional (2D) and three‐dimensional (3D) culture conditions. In a confluent 2D monolayer, 3T3BST‐2 cells compared to 3T3pCDNA cells resisted contact inhibition and had enhanced growth and survival that is associated with increased phosphorylated protein kinase AKT (T308), increased phosphorylated mitogen‐activated protein kinase (ERK1/2), and decreased pro‐apoptotic factor Bak. In a 3D spheroids model, 3T3BST‐2 spheroids were larger in area and had higher ATP content, an indication of anchorage‐independent survival. Suspended as single cells in a gel, 3T3BST‐2 cells also had enhanced survival, especially when arginylglycylaspartic acid (RGD) adhesive peptide motif was present. Suppressed contact inhibition and enhanced anchorage‐independent survival suggests that overexpression of BST‐2 contributes to malignant transformation of normal cells. Discerning the mechanisms of BST‐2 mediated oncogenesis will expand our understanding of events that transforms a normal cell into an unwanted cancerous cell, which may potentially form the foundation for the development of BST‐2‐targeted cancer therapies.Support or Funding Information1. Molecular and Cellular Pharmacology PhD program 2. CIE’s IMSD‐MERGE scholars program 3. Scholars in Biomedical Sciences program (SBMS) 4. Stony Brook University Cancer Center

MicroRNA-760 inhibits cell viability and migration through down-regulating BST2 in gastric cancer.

Gastric cancer is one of the most common types of carcinoma with a threat to global health. MicroRNA-760 (miR-760) was significantly down-regulated in the primary tumour of patients with advanced gastric cancer. However, the role of miR-760 in gastric cancer is still unclear. Herein, miR-760 was down-regulated in gastric cancer tissues. Moreover, miR-760 overexpression and knockdown were conducted in gastric cancer cells (MGC-803 and SGC-7901) in vitro. The in vitro functional assays proved that miR-760 overexpression reduced cell viability, cell cycle, migration and invasion, promoted apoptosis and suppressed MMP activity in MGC-803 cells. Conversely, miR-760 knockdown led to the opposite in SGC-7901 cells. Notably, bone marrow stromal antigen 2 (BST2) was verified as a target gene of miR-760. MiR-760 mimics down-regulated BST2 level in gastric cancer tissues and in MGC-803 cells, whereas miR-760 inhibitor up-regulated its level in SGC-7901 cells. MiR-760-regulated cell properties through reduction of BST2. In addition, miR-760 inhibited tumourigenesis in a nude mouse xenograft model in vivo. In conclusion, our results demonstrated that miR-760 exhibited a suppressive role in gastric cancer via inhibiting BST2, indicating that miR-760/BST2 axis may provide promising therapeutic target for gastric cancer.

Development and Characterization of the Shortest Anti-Adhesion Peptide Analogue of B49Mod1.

Inhibition of cancer cell adhesion is an effective approach to killing adherent cancer cells. B49 and its analog B49Mod1 peptides, derived from the extracellular domain (ECD) of bone marrow stromal antigen 2 (BST-2), display anti-adhesion activity on breast cancer cells. However, the minimal sequence required for this anti-adhesion activity is unknown. Here, we further characterized the anti-adhesion activity of B49Mod1. We show that the anti-adhesion activity of B49Mod1 may require cysteine-linked disulfide bond and that the peptide is susceptible to proteolytic deactivation. Using structure-activity relationship studies, we identified an 18-Mer sequence (B18) as the minimal peptide sequence mediating the anti-adhesion activity of B49Mod1. Atomistic molecular dynamic (MD) simulations reveal that B18 forms a stable complex with the ECD of BST-2 in aqueous solution. MD simulations further reveal that B18 may cause membrane defects that facilitates peptide translocation across the bilayer. Placement of four B18 chains as a transmembrane bundle results in water channel formation, indicating that B18 may impair membrane integrity and form pores. We hereby identify B18 as the minimal peptide sequence required for the anti-adhesion activity of B49Mod1 and provide atomistic insight into the interaction of B18 with BST-2 and the cell membrane.

IFNy Stimulation Induces Hematopoietic Stem Cell Homing and Niche Relocalization

Interferon gamma (IFNy) is a pro-inflammatory cytokine that is upregulated during chronic infections and chronic diseases, such as aplastic anemia, and has been associated with pancytopenia and diminished hematopoiesis. Studies have shown that IFNy negatively regulates hematopoietic stem cell (HSC) homeostasis by decreasing self-renewal and promoting terminal differentiation. The tight regulation of HSC homeostasis is dependent upon the bone marrow (BM) microenvironment, or BM niche. The BM niche is composed of a network of cell types that provide elaborate cell-cell interactions, cellular metabolites, transcriptional regulators, and local and distant humoral and neural signals that allow for hematopoietic homeostasis. In particular, CXCL12-abundant reticular (CAR) cells are vital to HSC maintenance, as depletion of CXCL12, or its receptor, leads to HSC depletion. However, the mechanism which IFNy activates HSCs and influences its interaction with the BM niche is unknown. We hypothesize that IFNy promotes HSC terminal differentiation and loss of quiescence by altering HSC interactions with the BM niche. To assess changes in HSC interactions with the BM niche upon IFNy stimulation, we performed intravital imaging using CXCL12 GFP reporter mice before and after administration of recombinant IFNy. We found that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. There was no change in distance with IFNy-receptor deficient HSCs, suggesting that movement away from the CAR cells was due to a cell autonomous IFNy-dependent mechanism. We performed gene expression analysis and transwell migration assays on HSCs from IFNy treated mice, and determined that there was no change in CXCL12 receptor (CXCR4) expression upon IFNy treatment, and IFNy did not alter migration towards CXCL12. These results suggest that HSC re-localization upon IFNy is independent of CXCL12 signaling. To explore the mechanism by which IFNy induces re-localization of HSCs, we first performed microarray analysis on HSCs from IFNy stimulated mice to assess what surface proteins were changed upon IFNy treatment. While there was no change in common HSC receptors thought to influence HSC homeostasis (cKit, Cdh2, Mpl, Itgb1, Itbg2, Itga4, and Itga1), we observed an increase in expression of bone marrow stromal antigen 2 (BST2). To explore the impact of BST2 on HSC homeostasis, quantification and proliferation analysis was performed on HSCs from Bst2-/- mice. Interestingly, Bst2-/- HSCs were significantly less proliferative and more abundant compared to controls. These studies suggest that BST2 may play a role in maintaining HSC homeostasis. The functional role of BST2 in cellular movement and adhesion has been studied in cancer. Increased BST2 expression has been associated with promoting the migration, adhesion and metastasis of various cancer cells. Since migration and adhesion is important for HSC homing, we assessed the effects of IFNy on HSC homing. Hematopoietic progenitors from IFNy-treated mice homed to the bone marrow with greater efficiency than PBS-treated controls, whereas progenitors from IFNy-receptor-deficientmice showed a decrease in homing. Additionally, WBM from IFNyR-/- had reduced engraftment than wildtype, consistent with a role for IFNy signaling in promoting HSC homing. The impact of BST2 on homing is currently being explored. In summary, we show that IFNy induces re-localization of HSCs away from quiescence-promoting CAR cells within the bone marrow niche via a mechanism that is independent of CXCL12 signaling. We further show that IFNy promotes HSC homing. The increased expression of BST2 on IFNy-stimulated HSCs appears to impact HSC proliferation and abundance in the bone marrow. Thus, BST2 may play a role in HSC activation and exit from quiescence. Expanding our understanding of the mechanism that drives HSC activation and terminal differentiation has important implications for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

Beyond Tethering the Viral Particles: Immunomodulatory Functions of Tetherin (BST-2).

Host response to viral infection is a highly regulated process involving engagement of various host factors, cytokines, chemokines, and stimulatory signals that pave the way for an antiviral immune response. The response is manifested in terms of viral sequestration, phagocytosis, and inhibition of genome replication, and, finally, if required, lymphocyte-mediated clearance of virally infected cells. During this process, cross-talk between viral and host factors can shape disease outcomes and immunopathology. Bone marrow stromal antigen 2 (BST-2), also know as tetherin, is induced by type I interferon produced in response to viral infections, as well as in certain cancers. BST-2 has been shown to be a host restriction factor of virus multiplication through its ability to physically tether budding virions and restrict viral spread. However, BST-2 has other roles in the host antiviral response. This review focuses on the diverse functions of BST-2 and its downstream signaling pathways in regulating host immune responses.