Anthrax Toxin Receptor 1 Research Articles

One Health Assessment of Bacillus anthracis Incidence and Detection in Anthrax-Endemic Areas of Pakistan.

Anthrax, a severe zoonotic disease, is infrequently reported in anthrax-endemic regions of Pakistan. Despite clinical reports indicating its presence, particularly cutaneous anthrax, there is insufficient laboratory evidence regarding disease occurrence and environmental persistence. The present study aimed to confirm Bacillus anthracis presence, accountable for animal mortality and human infection, while exploring environmental transmission factors. Between March 2019 and July 2021, a total of 19 outbreaks were documented. Of these, 11 affected sheep/goats in Zhob district and 8 affected cattle/sheep in Bajour Agency. Clinical signs suggestive of Bacillus anthracis outbreak were observed in 11 animals. Blood and swab samples were collected for confirmation. The study followed a One Health approach, analyzing animal, environmental (soil/plant), and human samples. Of the 19 outbreaks, 11 were confirmed positive for anthrax based on growth characteristics, colony morphology, and PCR. Soil and plant root samples from the outbreak areas were collected and analyzed microscopically and molecularly. Cutaneous anthrax was observed in six humans, and swab samples were taken from the lesions. Human serum samples (n = 156) were tested for IgG antibodies against PA toxin and quantitative analysis of anthrax toxin receptor 1 (ANTXR1). Bacillus anthracis was detected in 65 out of 570 (11.40%) soil samples and 19 out of 190 (10%) plant root samples from the outbreak areas. Four out of six human samples from cutaneous anthrax lesions tested positive for Bacillus anthracis. Human anthrax seroprevalence was found to be 11% and 9% in two districts, with the highest rates among butchers and meat consumers. The highest ANTXR1 levels were observed in butchers, followed by meat consumers, farm employees, meat vendors, veterinarians, and farm owners. These findings highlight the persistence of anthrax in the region and emphasize the potential public health risks.

ANTXR1 as a potential sensor of extracellular mechanical cues.

Cell adhesion molecules mediate cell-cell or cell-matrix interactions, some of which are mechanical sensors, such as integrins. Emerging evidence indicates that anthrax toxin receptor 1 (ANTXR1), a newly identified cell adhesion molecule, can also sense extracellular mechanical signals such as hydrostatic pressure and extracellular matrix (ECM) rigidity. ANTXR1 can interact with ECM through connecting intracellular cytoskeleton and ECM molecules (just like integrins) to regulate numerous biological processes, such as cell adhesion, cell migration or ECM homeostasis. Although with high structural similarity to integrins, its functions and downstream signal transduction are independent from those of integrins. In this perspective, based on existing evidence in literature, we analyzed the structural and functional evidence that ANTXR1 can act as a potential sensor for extracellular mechanical cues. To our knowledge, this is the first in-depth overview of ANTXR1 from the perspective of mechanobiology. STATEMENT OF SIGNIFICANCE: An overview of ANTXR1 from the perspective of mechanobiology; An analysis of mechanical sensitivity of ANTXR1 in structure and function; A summary of existing evidence of ANTXR1 as a potential mechanosensor.

ANTXR1 Regulates Erythroid Cell Proliferation and Differentiation through wnt/β-Catenin Signaling Pathway In Vitro and in Hematopoietic Stem Cell.

Erythropoiesis is a highly complex and sophisticated multistage process regulated by many transcription factors, as well as noncoding RNAs. Anthrax toxin receptor 1 (ANTXR1) is a type I transmembrane protein that binds the anthrax toxin ligands and mediates the entry of its toxic part into cells. It also functions as a receptor for the Protective antigen (PA) of anthrax toxin, and mediates the entry of Edema factor (EF) and Lethal factor (LF) into the cytoplasm of target cells and exerts their toxicity. Previous research has shown that ANTXR1 inhibits the expression of γ-globin during the differentiation of erythroid cells. However, the effect on erythropoiesis from a cellular perspective has not been fully determined. This study examined the role of ANTXR1 on erythropoiesis using K562 and HUDEP-2 cell lines as well as cord blood CD34+ cells. Our study has shown that overexpression of ANTXR1 can positively regulate erythrocyte proliferation, as well as inhibit GATA1 and ALAS2 expression, differentiation, and apoptosis in K562 cells and hematopoietic stem cells. ANTXR1 knockdown inhibited proliferation, promoted GATA1 and ALAS2 expression, accelerated erythrocyte differentiation and apoptosis, and promoted erythrocyte maturation. Our study also showed that ANTXR1 may regulate the proliferation and differentiation of hematopoietic cells, though the Wnt/β-catenin pathway, which may help to establish a possible therapeutic target for the treatment of blood disorders.

Disruption of anthrax toxin receptor 1 in pigs leads to a rare disease phenotype and protection from senecavirus A infection

Senecavirus A (SVA) is a cause of vesicular disease in pigs, and infection rates are rising within the swine industry. Recently, anthrax toxin receptor 1 (ANTXR1) was revealed as the receptor for SVA in human cells. Herein, the role of ANTXR1 as a receptor for SVA in pigs was investigated by CRISPR/Cas9 genome editing. Strikingly, ANTXR1 knockout (KO) pigs exhibited features consistent with the rare disease, GAPO syndrome, in humans. Fibroblasts from wild type (WT) pigs supported replication of SVA; whereas, fibroblasts from KO pigs were resistant to infection. During an SVA challenge, clinical symptoms, including vesicular lesions, and circulating viremia were present in infected WT pigs but were absent in KO pigs. Additional ANTXR1-edited piglets were generated that were homozygous for an in-frame (IF) mutation. While IF pigs presented a GAPO phenotype similar to the KO pigs, fibroblasts showed mild infection, and circulating SVA nucleic acid was decreased in IF compared to WT pigs. Thus, this new ANTXR1 mutation resulted in decreased permissiveness of SVA in pigs. Overall, genetic disruption of ANTXR1 in pigs provides a unique model for GAPO syndrome and prevents circulating SVA infection and clinical symptoms, confirming that ANTXR1 acts as a receptor for the virus.

MiR-381-3p Involves in Glioma Progression by Suppressing Tumor-Promoter Factor ANTXR1.

Accumulating studies revealed association between development of glioma and miRNA dysregulation. A case in point is miR-381-3p, but its mechanism in glioma is unclear yet. In this work, we confirmed that overexpressed miR-381-3p repressed biological functions of glioma cells. Additionally, we also discovered that upregulated anthrax toxin receptor 1 (ANTXR1) was negatively mediated by miR-381-3p. We further proved that miR-381-3p-targeted ANTXR1 was able to counteract the suppression of miR-381-3p on biological functions of glioma. We concluded that miR-381-3p and ANTXR1 were both important factors in modulating glioma progression. miR-381-3p/ANTXR1 axis is expected to be a molecular target for glioma.

ANTXR1 as a potential prognostic biomarker for hepatitis B virus-related hepatocellular carcinoma identified by a weighted gene correlation network analysis.

With high incidence and mortality rates, hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors worldwide. Chronic hepatitis B virus (HBV) infection is a leading cause of HCC, especially for Asians and blacks. However, the molecular mechanisms underlying HBV-related HCC are unclear. This study sought to identify novel prognostic biomarkers and explore the potential pathogenesis of HBV-related HCC. The gene expression profiles and corresponding clinical information of HCC from The Cancer Genome Atlas Liver Hepatocellular Carcinoma data set were analyzed by a weighted gene co-expression network analysis. Correlations between the co-expression modules and clinical traits were calculated. Next, key modules associated with HBV infection were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted for the genes in the key modules. The hub genes were identified based on the protein-protein interaction (PPI) network via the Cytoscape. Finally, an overall survival (OS) analysis was performed. The two modules (i.e., the brown and yellow modules) most relevant to HBV infection were constructed. A functional enrichment analysis revealed that the genes in the two modules were mainly enriched in HCC-related pathways, such as the phosphatidylinositol-3-kinase and protein kinase B signaling pathway, focal adhesion, human papillomavirus infection, the Rap1 signaling pathway, and the cyclic guanosine monophosphate-dependent protein kinase (cGMP-PKG) signaling pathway. Ten hub genes [i.e., COL3A1, ANTXR1, COL14A1, THBS2, ADAMTS2, AEBP1, PRELP, EMILIN1, DCN and PODN] in the brown module, and 10 hub genes [i.e., USP34, SEC24C, ZNF770, STAG1, TSTD2, PKD1P6, CCNK, GFT2I, NT5C2 and SMG6] in the yellow module were identified. Among the hub genes, ANTXR1 (Anthrax-toxin receptor 1) was significantly correlated with HBV-related HCC patients' OS. ANTXR1 represents a potential therapeutic target for HBV-related HCC. This study offers novel insights into the molecular mechanisms of HBV-induced tumorigenesis, which needs to be further validated by basic experiments and large-scale cohort studies.

N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection.

Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.

Latest therapeutic target for gastric cancer: Anthrax toxin receptor 1.

Anthrax toxin receptor 1 (ANTXR1), also known as tumor endothelial marker 8, is a highly conserved cell surface protein overexpressed in tumor-infiltrating vessels. It was first found in vascular endothelial cells of human colorectal cancer. Although our understanding of its physiological function is limited, it has been found that ANTXR1 binds collagen and promotes migration of endothelial cells in vitro. ANTXR1 is upregulated in vessels of different tumor types in mice and humans, and is also expressed by tumor cells themselves in some tumors, such as gastric, lung, intestinal and breast cancer. Developmental angiogenesis and wound healing were not disturbed in ANTXR1 knockout mice, but compared with wild-type mice, growth of melanoma was impaired after ANTXR1 knockout, indicating that host-derived ANTXR1 can promote tumor growth on the basis of immune activity. Previous studies have shown that ANTXR1 vaccines or sublethal doses of anthrax toxin can inhibit angiogenesis, slow tumor growth and prolong survival. These studies suggest that ANTXR1 is necessary for tumor rather than physiological angiogenesis. It has been found that ANTXR1 plays an important role in tumor angiogenesisas well as in the growth and metastasis of many kinds of tumors. This article reviews the physiological function of ANTXR1 and its role in different kinds of cancer.

Infectious recombinant Senecavirus A expressing novel reporter proteins.

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. The construction of SVA virus carrying foreign reporter gene provides a powerful tool in virus research. However, it is often fraught with rescuing a recombinant picornavirus harboring a foreign gene or maintaining the stability of foreign gene in the virus genome. Here, we successfully generated recombinant SVA GD05/2017 viruses (V-GD05-clone) expressing the green fluorescent protein (iLOV), red fluorescent protein (RFP), or NanoLuc luciferase (Nluc). These recombinant viruses have comparable growth kinetics to the parental virus. Genetic stability analysis indicated that V-GD05-iLOV was highly stable in retaining iLOV gene for more than 10 passages, while V-GD05-RFP and V-GD05-Nluc lost the foreign genes in five passages. In addition, high-intensity fluorescent signals were found in the V-GD05-RFP- and V-GD05-iLOV-infected cells by fluorescence observation and flow cytometry analysis, and the luciferase activity assay could quantitatively monitor the replication of V-GD05-Nluc. In order to identify the porcine cell receptor for SVA, anthrax toxin receptor 1 (ANTXR1) was knocked out or overexpressed in the ST-R cells. The ANTXR1 knock-out cells lost the ability for SVA infection, while overexpression of ANTXR1 significantly increased the cell permissivity. These results confirmed that ANTXR1 was the receptor for SVA to invade porcine cells as reported in the human cells. Overall, this study suggests that these SVA reporter viruses will be useful tools in elucidating virus pathogenesis and developing control measures.Key points • We successfully generated SVA viruses expressing the iLOV, RFP, or Nluc. • The iLOV was genetically stable in the V-GD05-iLOV genome over ten passages. • ANTXR1 was the receptor for SVA to invade porcine cells. Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-021-11181-6.

ANTXR1 Is a Prognostic Biomarker and Correlates With Stromal and Immune Cell Infiltration in Gastric Cancer.

Gastric cancer (GC) is a complex and heterogeneous disease, making it difficult to ascertain the optimal therapeutic approach for individual GC patients. Stromal and immune cell infiltration in GC has a strong correlation with clinical outcomes; however, the underlying mechanisms that drive immunosuppression remain vastly undiscovered. Recent studies validated that anthrax toxin receptor 1 (ANTXR1) is aberrantly expressed in several cancers and holds promise as a new therapeutic target for cancer. However, its immunological roles in GC are still unclear. Here, we show that we identify the distinct stromal and immune cell infiltration in GC between the high and low ANTXR1 expression group by analyzing genomic data. Clinically, ANTXR1 is highly expressed in GC and correlates with adverse clinicopathological characteristics. Additionally, high ANTXR1 expression is linked to markedly poor clinical outcomes and resistance to chemotherapy, whereas the low ANTXR1 expression group is correlated with better outcomes and response to chemotherapy in GC patients. We further revealed the differential landscape of somatic tumor mutation burden (TMB) between the two groups and observed that patients with high ANTXR1 expression suffered from a lower TMB, potentially leading to less sensitivity to checkpoint therapy. Molecularly, results displayed that ANTXR1 is an immunosuppressive element, which may perform its function via promoting the secretion of immunosuppressive factors that play a significant role in modulating tumor-associated fibroblast transformation, M2 macrophage polarization, and T cell exhaustion. Gene set enrichment analysis revealed that cancer-related pathways including epithelial-to-mesenchymal transition, focal adhesion, and transforming growth factor-β (TGF-β) signaling pathways were enriched in high ANTXR1 expression tumors. Our work suggests that ANTXR1 could not only serve as a valuable prognostic biomarker in GC but also be deemed as a potential immunotherapeutic target and useful biomarker of sensitivity to chemotherapy.

Anthrax toxin receptor 1/tumor endothelial marker 8 promotes gastric cancer progression through activation of the PI3K/AKT/mTOR signaling pathway.

Anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein, is one of the receptors that facilitates the entrance of anthrax toxin into cells. Previous studies have confirmed the pivotal role of ANTXR1 in progression and tumorigenesis of diverse cancer types. However, the biological function of ANTXR1 in gastric cancer (GC) is still unknown. The present study aimed to investigate the role of ANTXR1 in GC and illuminate the potential molecular mechanisms. Bioinformatics analysis found that ANTXR1 expression was significantly upregulated in GC tissue and its overexpression was associated with poor prognosis of GC patients. Moreover, we confirmed the upregulation of ANTXR1 in GC cell lines and GC tissue by quantitative PCR, western blot analysis, and immunohistochemical analysis. Additionally, high protein expression level of ANTXR1 was positively associated with several clinicopathological parameters in GC patients. In our study, a series of in vitro and in vivo assays were undertaken through strategies of loss/gain‐of‐function and rescue assays. Consequently, our results indicated that ANTXR1 induced proliferation, cell cycle progression, invasion and migration, and tumorigenicity and induced suppressed apoptosis in GC. Mechanistic investigation indicated that ANTXR1 exerted its promoting effects on GC through activation of the PI3K/AKT/mTOR signaling pathway. In conclusion, our findings suggested that ANTXR1 plays a crucial role in the development and progression of GC and could serve as a novel prognostic biomarker and potential therapeutic target for GC.

The Anthrax Toxin Receptor 1 (ANTXR1) Is Enriched in Pancreatic Cancer Stem Cells Derived from Primary Tumor Cultures

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related mortality. Cancer stem cells (CSCs) have been shown to be the drivers of pancreatic tumor growth, metastasis, and chemoresistance, but our understanding of these cells is still limited by our inability to efficiently identify and isolate them. While a number of markers capable of identifying pancreatic CSCs (PaCSCs) have been discovered since 2007, there is no doubt that more markers are still needed. The anthrax toxin receptor 1 (ANTXR1) was identified as a functional biomarker of triple-negative breast CSCs, and PDAC patients stratified based on ANTXR1 expression levels showed increased mortality and enrichment of pathways known to be necessary for CSC biology, including TGF-β, NOTCH, Wnt/β-catenin, and IL-6/JAK/STAT3 signaling and epithelial to mesenchymal transition, suggesting that ANTXR1 may represent a putative PaCSC marker. In this study, we show that ANTXR1+ cells are not only detectable across a panel of 7 PDAC patient-derived xenograft primary cultures but ANTXR1 expression significantly increased in CSC-enriched 3D sphere cultures. Importantly, ANTXR1+ cells also coexpressed other known PaCSC markers such as CD44, CD133, and autofluorescence, and ANTXR1+ cells displayed enhanced CSC functional and molecular properties, including increased self-renewal and expression of pluripotency-associated genes, compared to ANTXR1− cells. Thus, this study validates ANTXR1 as a new PaCSC marker and we propose its use in identifying CSCs in this tumor type and its exploitation in the development of CSC-targeted therapies for PDAC.

Role of ANTXR1 in the regulation of RANKL-induced osteoclast differentiation and function

Anthrax toxin receptor 1 (ANTXR1) is a transmembrane protein with an extracellular domain which is deeply associated with the process of bone formation and plays an important role in angiogenesis. However, there have been no reports investigating the effects of ANTXR1 on bone metabolism mediated by the two types of bone cells, osteoclasts, and osteoblasts. The aim of this study is to reveal the role of ANTXR1 in the differentiation and function of osteoclasts and osteoblasts. We found that ANTXR1 positively regulated the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and bone resorption with no effects on osteoblast differentiation by performing gain- and loss-of-function studies. During ANTXR1-mediated regulation of osteoclastogenesis, phosphorylation of early signal transducers such as c-Jun N-terminal kinase (JNK), Akt, inhibitor of kappa B (IκB), and phospholipase C gamma 2 (PLCγ2) was affected, which in turn altered the mRNA and protein levels of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In addition, genetic manipulation of ANTXR1 in bone marrow macrophages (BMMs) modulated the capillary-like tube formation in HUVECs via secretion of two angiogenic factors, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A). These results elucidated the importance of ANTXR1 in osteoclast differentiation and functional activity, as well as, osteoclast-mediated angiogenesis of endothelial cells. Taken together, we propose that ANTXR1 might be a promising candidate for gene therapy for bone metabolic diseases and further, might potentially serve as an important biomarker in the field of bone metastasis associated with vascularization.

Seneca Valley virus attachment and uncoating mediated by its receptor anthrax toxin receptor 1

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. SVV mediates cell entry by attachment to the receptor anthrax toxin receptor 1 (ANTXR1). Here we determine atomic structures of mature SVV particles alone and in complex with ANTXR1 in both neutral and acidic conditions, as well as empty "spent" particles in complex with ANTXR1 in acidic conditions by cryoelectron microscopy. SVV engages ANTXR1 mainly by the VP2 DF and VP1 CD loops, leading to structural changes in the VP1 GH loop and VP3 GH loop, which attenuate interprotomer interactions and destabilize the capsid assembly. Despite lying on the edge of the attachment site, VP2 D146 interacts with the metal ion in ANTXR1 and is required for cell entry. Though the individual substitution of most interacting residues abolishes receptor binding and virus propagation, a serine-to-alanine mutation at VP2 S177 significantly increases SVV proliferation. Acidification of the SVV-ANTXR1 complex results in a major reconfiguration of the pentameric capsid assemblies, which rotate ∼20° around the icosahedral fivefold axes to form a previously uncharacterized spent particle resembling a potential uncoating intermediate with remarkable perforations at both two- and threefold axes. These structures provide high-resolution snapshots of SVV entry, highlighting opportunities for anticancer therapeutic optimization.

Structural basis for anthrax toxin receptor 1 recognition by Seneca Valley Virus

Recently, the use of oncolytic viruses in cancer therapy has become a realistic therapeutic option. Seneca Valley Virus (SVV) is a newly discovered picornavirus, which has earned a significant reputation as a potent oncolytic agent. Anthrax toxin receptor 1 (ANTXR1), one of the cellular receptors for the protective antigen secreted by Bacillus anthracis, has been identified as the high-affinity cellular receptor for SVV. Here, we report the structure of the SVV-ANTXR1 complex determined by single-particle cryo-electron microscopy analysis at near-atomic resolution. This is an example of a shared receptor structure between a mammalian virus and a bacterial toxin. Our structure shows that ANTXR1 decorates the outer surface of the SVV capsid and interacts with the surface-exposed BC loop and loop II of VP1, "the puff" of VP2 and "the knob" of VP3. Comparison of the receptor-bound capsid structure with the native capsid structure reveals that receptor binding induces minor conformational changes in SVV capsid structure, suggesting the role of ANTXR1 as an attachment receptor. Furthermore, our results demonstrate that the capsid footprint on the receptor is not conserved in anthrax toxin receptor 2 (ANTXR2), thereby providing a molecular mechanism for explaining the exquisite selectivity of SVV for ANTXR1.

Molecular Adjuvants Based on Plasmids Encoding Protein Aggregation Domains Affect Bone Marrow Niche Homeostasis.

Background:During last years, DNA vaccine immunogenicity has been optimized by the employment of co-stimulatory molecules and molecular adjuvants. It has been reported that plasmid (pATRex), encompassing the DNA sequence for the von Willebrand A (vWA/A) domain of the An-thrax Toxin Receptor-1 (ANTXR-1, alias TEM8, Tumor Endothelial Marker 8), acts as strong immune adjuvant by inducing formation of insoluble intracellular aggregates. Markedly, we faced with upsetting findings regarding the safety of pATRex as adjuvant since the aggregosome formation prompted to os-teopenia in mice.Objective:The present study provides additional evidences about the proteinaceous adjuvants action within bone marrow and questioned regarding the self-aggregation protein adjuvants immunotoxicity on marrow niches.Methods & Results:Using histological, biochemical and proteomic assays we shed light on pATRex effects within bone marrow niche and specifically we evidenced an aplastic-like bone marrow with dis-rupted cytokine/chemokine production.Conclusion:The above findings provide compelling support to the thesis that adjuvants based on plas-mids encoding protein aggregation domains disrupt the physiological features of the bone marrow ele-ments.

Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus.

Seneca Valley virus (SVV) is an oncolytic picornavirus with selective tropism for neuroendocrine cancers. It has shown promise as a cancer therapeutic in preclinical studies and early-phase clinical trials. Here, we have identified anthrax toxin receptor 1 (ANTXR1) as the receptor for SVV using genome-wide loss-of-function screens. ANTXR1 is necessary for permissivity in vitro and in vivo. However, robust SVV replication requires an additional innate immune defect. We found that SVV interacts directly and specifically with ANTXR1, that this interaction is required for SVV binding to permissive cells, and that ANTXR1 expression is necessary and sufficient for infection in cell lines with decreased expression of antiviral IFN genes at baseline. Finally, we identified the region of the SVV capsid that is responsible for receptor recognition using cryoelectron microscopy of the SVV-ANTXR1-Fc complex. These studies identify ANTXR1, a class of receptor that is shared by a mammalian virus and a bacterial toxin, as the cellular receptor for SVV.

MicroRNA-493 suppresses hepatocellular carcinoma tumorigenesis through down-regulation of anthrax toxin receptor 1 (ANTXR1) and R-Spondin 2 (RSPO2)

Hepatocellular carcinoma (HCC) is known as a highly prevalent cancer with a poor prognosis and short survival time, despite intensive research and clinical efforts. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of hepatocellular carcinoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we report that the expression of microRNA-493 (miR-493) is decreased in HCC cell lines and in tumor tissues. Overexpression of miR-493 in HCC cells dramatically inhibited cell proliferation and colony-formation in vitro and inhibited tumor formation of HCC cell xenografts in vivo. miR-493 also suppressed cell migration and invasion in HCC cell lines. Novel targets ANTXR1 and RSPO2 were confirmed to be suppressed by miR-493 directly, and overexpression of ANTXR1 and RSPO2 could restore tumorigenesis in miR-493 treated HCC cell. Moreover, Wnt/β-catenin signaling pathway, which was reported to be activated by ANTXR1 and RSPO2, was also inhibited by miR-493 overexpression and might be involved in anti-tumor function of miR-493. These findings suggest that miR-493 acts as a negative regulator in hepatocellular carcinoma progression and may be a potential therapeutic target for HCC.

Abstract 4356: Identification of the anthrax toxin receptor (ANTXR1) as the high affinity cellular receptor for Seneca Valley Virus (SVV)

Abstract Small cell lung cancer (SCLC) is an extremely aggressive and almost universally lethal form of lung cancer, which accounts for approximately 15% of all lung cancer diagnoses annually. Novel and more effective therapies are desperately needed for SCLC, as the standard treatment has not improved in almost three decades. Recently, oncolytic viruses have shown strong anti-cancer efficacies in tumors with high levels of selectivity for cancer cells. The oncolytic picornavirus, Seneca Valley Virus (SVV), has previously been shown to infect and lyse cancer cells with neuroendocrine features, notably SCLC and pediatric brain tumors. Pre-clinical mouse models and early phase clinical trials have confirmed the ability of SVV to home directly to the tumor through the vasculature, specifically infect tumor cells without affecting normal cells, and replicate to high viral titers intratumorally even after the production of neutralizing antibodies. However, the clinical development of SVV has been hindered by the inability to identify patients who would benefit from SVV virotherapy because essential host proteins for viral entry, including the cellular receptor, have not been characterized. Using genome wide loss-of-function CRISPR screens in the SCLC cell line H446 and haploid cell line HAP1, we identified the anthrax toxin receptor 1 (ANTXR1) as an essential host protein for SVV. Interestingly, ANTXR1 is also one of the receptors for the toxin of Bacillus anthracis and has been previously described as being upregulated in tumor endothelial cells. Secondary screens performed in both cell lines confirmed gene knockout of ANTXR1 conferred resistance to SVV. We established that in multiple permissive SCLC and pediatric cancer cell lines that loss of ANTXR1 protein expression using CRISPR gene knockout leads to a loss of SVV permissivity. Re-expression of ANTXR1 in ANTXR1 knockout cell lines was sufficient to rescue SVV permissivity. Furthermore, the expression of the ANTXR1 protein in two non-permissive SCLC cell lines led to a conversion in SVV permissivity. A direct and high affinity interaction was confirmed between SVV and ANTXR1 using co-immunoprecipitation assays. Additionally, we discovered that ANTXR1 is the major binding determinant for SVV on cells using a binding assay with fluorescently labeled SVV. Lastly we determined using previously published gene expression data from the Cancer Cell Line Encyclopedia (CCLE) that non-permissive lines which do express ANTXR1 can be explained by the presence of an intact and active innate immune response pathway. These findings identify ANTXR1 as the high-affinity cellular receptor for SVV. Furthermore, these data identify a predictive biomarker for SVV permissivity that could lead to defined selection criteria for subsequent clinical trials testing SVV in both SCLC and pediatric brain cancer patients. Citation Format: Linde A. Miles, J.T. Poirier, Charles M. Rudin. Identification of the anthrax toxin receptor (ANTXR1) as the high affinity cellular receptor for Seneca Valley Virus (SVV). [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 4356.

Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis

It is well known that angiogenesis is linked to fibrotic processes in fibroproliferative diseases, but insights into pathophysiological processes are limited, due to lack of understanding of molecular mechanisms controlling endothelial and fibroblastic homeostasis. We demonstrate here that the matrix receptor anthrax toxin receptor 1 (ANTXR1), also known as tumor endothelial marker 8 (TEM8), is an essential component of these mechanisms. Loss of TEM8 function in mice causes reduced synthesis of endothelial basement membrane components and hyperproliferative and leaky blood vessels in skin. In addition, endothelial cell alterations in mutants are almost identical to those of endothelial cells in infantile hemangioma lesions, including activated VEGF receptor signaling in endothelial cells, increased expression of the downstream targets VEGF and CXCL12, and increased numbers of macrophages and mast cells. In contrast, loss of TEM8 in fibroblasts leads to increased rates of synthesis of fiber-forming collagens, resulting in progressive fibrosis in skin and other organs. Compromised interactions between TEM8-deficient endothelial and fibroblastic cells cause dramatic reduction in the activity of the matrix-degrading enzyme MMP2. In addition to insights into mechanisms of connective tissue homeostasis, our data provide molecular explanations for vascular and connective tissue abnormalities in GAPO syndrome, caused by loss-of-function mutations in ANTXR1. Furthermore, the loss of MMP2 activity suggests that fibrotic skin abnormalities in GAPO syndrome are, in part, the consequence of pathophysiological mechanisms underlying syndromes (NAO, Torg and Winchester) with multicentric skin nodulosis and osteolysis caused by homozygous loss-of-function mutations in MMP2.