Coxsackievirus and Adenovirus Receptor (CXADR): Recent Findings and Its Role and Regulation in Spermatogenesis.

Coxsackievirus and adenovirus receptor (CXADR) belongs to immunoglobulin superfamily of cell adhesion molecules. It expresses in most tissues, but displays unique and indispensable functions in some tissues such as heart and testis. CXADR is a multifunctional protein that can serve as a viral receptor, a junction structural protein and a signalling molecule. Thus, it exerts a wide range of functions such as facilitating leukocyte transmigration, regulating barrier function and cell adhesion, promoting EMT transition, and mediating spermatogenesis. This review aims to provide an overview and highlights some recent findings on CXADR in the field with emphasis on studies in the testis, upon which future studies can be designed to delineate the roles and regulation of CXADR in spermatogenesis.

JAML promotes CD8 and γδ T cell antitumor immunity and is a novel target for cancer immunotherapy.

T cells are critical mediators of antitumor immunity and a major target for cancer immunotherapy. Antibody blockade of inhibitory receptors such as PD-1 can partially restore the activity of tumor-infiltrating lymphocytes (TILs). However, the activation signals required to promote TIL responses are less well characterized. Here we show that the antitumor activity of CD8 and γδ TIL is supported by interactions between junctional adhesion molecule-like protein (JAML) on T cells and its ligand coxsackie and adenovirus receptor (CXADR) within tumor tissue. Loss of JAML through knockout in mice resulted in accelerated tumor growth that was associated with an impaired γδ TIL response and increased CD8 TIL dysfunction. In mouse tumor models, therapeutic treatment with an agonistic anti-JAML antibody inhibited tumor growth, improved γδ TIL activation, decreased markers of CD8 TIL dysfunction, and significantly improved response to anti-PD-1 checkpoint blockade. Thus, JAML represents a novel therapeutic target to enhance both CD8 and γδ TIL immunity.

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation.

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

Enhanced expression of coxsackievirus and adenovirus receptor in lipopolysaccharide-induced inflammatory macrophages is through TRIF-dependent innate immunity pathway.

AIMS: Inflammatory macrophages have been proposed as a therapeutic target for joint disorders caused by inflammation. This study aimed to investigate the expression and regulation of coxsackievirus-adenovirus receptor (CAR) in lipopolysaccharide (LPS)-stimulated inflammatory macrophages whereby to evaluate the feasibility of virus-directed enzyme prodrug therapy (VDEPT). MAIN METHODS: Macrophage cell lines (RAW264.7 and J774A.1) and primary macrophage cells derived from rat spleen were used to evaluate the expression of CAR protein or CAR mRNA. Specific inhibitors for TLR4 pathway were used to investigate the regulation of CAR expression. CAR expression in rat joints was documented by immunohistochemistry. Conditionally replicating adenovirus, CRAd-EGFP(PS1217L) or CRAd-NTR(PS1217H6), and non-replicating adenovirus CTL102 were used to transduce genes for enhanced green fluorescent protein (EGFP) or nitroreductase (NTR), respectively. The expression of EGFP, NTR, and the toxicity induced by CB1954 activation were evaluated. KEY FINDINGS: The in vitro experiments revealed that CAR upregulation was mediated through the TLR4/TRIF/IRF3 pathway in LPS-stimulated inflammatory macrophage RAW264.7 and J774A.1 cells. The inflammatory RAW264.7 cells upregulated CAR expression following LPS stimulation, leading to higher infectability, increased NTR expression, and enhanced sensitization to CB1954. In animal experiments, the induction of CAR expression was observed in the CD68-expressing primary macrophages and in the CD68-expressing macrophages within joints following LPS stimulation. SIGNIFICANCE: In conclusion, we report an enhanced CAR expression in inflammatory macrophages in vitro and in vivo through the immune response elicited by LPS. Thus, the TLR4/TRIF/IRF3 pathway of macrophages, when activated, could facilitate the therapeutic application of adenovirus-mediated VDEPT.

CXADR-like membrane protein protects against heart injury by preventing excessive pyroptosis after myocardial infarction.

Myocardial infarction (MI) results in cardiomyocyte death and ultimately leads to heart failure. Pyroptosis is a type of the inflammatory programmed cell death that has been found in various diseased tissues. However, the role of pyroptosis in MI heart remains unknown. Here, we showed that CXADR-like membrane protein (CLMP) was involved in pyroptosis in the mouse MI heart. Our data showed that CLMP was strongly expressed in fibroblasts of the infarcted mouse hearts. The Clmp+/- mice showed more serious myocardial fibrosis and ventricular dysfunction post-MI than wild-type (Clmp+/+ ) mice, indicating a protective effect of the fibroblast-expressed CLMP against MI-induced heart damage. Transcriptome analyses by RNA sequencing indicated that Il-1ß mRNA was significantly increased in the MI heart of Clmp+/- mouse, which indicated a more serious inflammatory response. Meanwhile, cleaved caspase-1 and Gasdermin D were significantly increased in the Clmp+/- MI heart, which demonstrated enhanced pyroptosis in the Clmp knockdown heart. Further analysis revealed that the pyroptosis mainly occurred in cardiac fibroblasts (CFs). Compared to wild-type fibroblasts, Clmp+/- CFs showed more serious pyroptosis and inflammatory after LPS plus nigericin treatment. Collectively, our results indicate that CLMP participates in the pyroptotic and inflammatory response of CFs in MI heart. We have provided a novel pyroptotic insight into the ischaemic heart, which might hold substantial potential for the treatment of MI.

Adenovirus Receptor Expression in Cancer and Its Multifaceted Role in Oncolytic Adenovirus Therapy.

Oncolytic adenovirus therapy is believed to be a promising way to treat cancer patients. To be able to target tumor cells with an oncolytic adenovirus, expression of the adenovirus receptor on the tumor cell is essential. Different adenovirus types bind to different receptors on the cell, of which the expression can vary between tumor types. Pre-existing neutralizing immunity to human adenovirus species C type 5 (HAdV-C5) has hampered its therapeutic efficacy in clinical trials, hence several adenoviral vectors from different species are currently being developed as a means to evade pre-existing immunity. Therefore, knowledge on the expression of appropriate adenovirus receptors on tumor cells is important. This could aid in determining which tumor types would benefit most from treatment with a certain oncolytic adenovirus type. This review provides an overview of the known receptors for human adenoviruses and how their expression on tumor cells might be differentially regulated compared to healthy tissue, before and after standardized anticancer treatments. Mechanisms behind the up- or downregulation of adenovirus receptor expression are discussed, which could be used to find new targets for combination therapy to enhance the efficacy of oncolytic adenovirus therapy. Additionally, the utility of the adenovirus receptors in oncolytic virotherapy is examined, including their role in viral spread, which might even surpass their function as primary entry receptors. Finally, future directions are offered regarding the selection of adenovirus types to be used in oncolytic adenovirus therapy in the fight against cancer.

Naturally occurring variants in the transmembrane and cytoplasmic domains of the human Coxsackie- and adenovirus receptor have no impact on virus internalisation.

The Coxsackie- and adenovirus receptor (CAR) mediates homophilic cell-cell contacts and susceptibility to both human pathogenic viruses through its membrane-distal immunoglobulin domain. In the present study, we screened five missense variants of the human CAR gene for their influence on adenovector or Coxsackievirus entry into Chinese hamster ovary cells. The CAR variants facilitated virus internalisation to a similar extent as wild type CAR. This underlines CAR's presumed invariance and essential physiological role in embryogenesis.

The coxsackievirus and adenovirus receptor: virological and biological beauty.

The coxsackievirus and adenovirus receptor (CAR) is an essential multifunctional cellular protein that is only beginning to be understood. CAR serves as a receptor for many adenoviruses, human group B coxsackieviruses, swine vesicular disease virus, and possibly other viruses. While named for its function as a viral receptor, CAR is also involved in cell adhesion, immune cell activation, synaptic transmission, and signaling. Knockout mouse models were first to identify some of these biological functions; however, tissue-specific model systems have shed light on the complexity of different CAR isoforms and their specific activities. Many of these functions are mediated by the large number of interacting proteins described so far, and several new putative interactions have recently been discovered. As antiviral and gene therapy strategies that target CAR continue to emerge, future work poised to understand the biological implications of manipulating CAR in vivo is critical.

Congenital short bowel syndrome: systematic review of a rare condition.

BACKGROUND: Congenital short bowel syndrome (CSBS) is a rare gastrointestinal disorder caused by intrauterine reduction of small bowel length whose etiology is still unknown. Chronic diarrhea, vomiting, and failure to thrive are the most important complications, arising from less absorptive intestinal surface. This review examines clinical features and outcomes of CSBS patients. METHODS: A PubMed and EMBASE research on CSBS was performed. Inclusion criterion was congenital short bowel diagnosis in a range of ages between 33 weeks of gestational age and 15 years old (IQR 38 days). Exclusion criteria were history of atresia of any part of the gastrointestinal tract and extensive surgical bowel resections. Qualitative and quantitative variables were collected and analyzed. Data were expressed in mean and IQR. RESULTS: Sixty-one patients were identified (38 males, 23 females) from 1969 to date. Mean bowel length was 58.24 cm (IQR 37.5). Malrotation of the midgut was seen in 98.4% of cases. Our data showed an interesting trend in improving the survival rate of these patients (from 28.5% before 2008 to 75% in the period after 2008). Sepsis was the most frequent cause of death reported (57.9%). Interestingly, 18 patients were genetically analyzed, finding mutations either in FLNA gene (38.8%) or in CLMP gene (61.1%). CONCLUSIONS: CSBS is a condition that seems to be related to an autosomal recessive (CLMP) or an X linked (FLNA) type of inheritance. Advance in medical management seems to have improved survival of these children in recent years. Further genetic studies can better understand the causes of this disease aiming to create personalized treatment. TYPE OF STUDY: Systematic review. LEVEL OF EVIDENCE: Level IV.

Identification of novel human adenovirus candidates using the coxsackievirus and adenovirus receptor for cell entry.

BACKGROUND: There are over 100 known human adenovirus (HAdV) types, which are able to cause a broad variety of different self-limiting but also lethal diseases especially in immunocompromised patients. Only limited information about the pathogenesis and biology of the majority of these virus types is available. In the present study, we performed a systematic screen for coxsackievirus and adenovirus receptor (CAR)-usage of a large spectrum of HAdV types. METHODS: To study receptor usage we utilized a recombinant HAdV library containing HAdV genomes tagged with a luciferase and GFP encoding transgene. We infected CHO-CAR cells stably expressing the CAR receptor and to much information with tagged viruses (HAdV3, 14, 16, 50, 10, 24, 27, 37 and 69) and measured luciferase expression levels 26 and for some viruses (AdV10, - 24 and - 27) 52 h post-infection. As positive control, we applied human adenovirus type 5 (HAdV5) known to use the CAR receptor for cell entry. For viruses replication studies on genome level we applied digital PCR. RESULTS: Infection of CHO-CAR and CHO-K1 cells at various virus particle numbers per cell (vpc) revealed that HAdV10, 24, and 27 showed similar or decreased luciferase expression levels in the presence of CAR. In contrast, HAdV3, 14, 16, 50, 37 and 69 resulted in increased luciferase expression levels in our initial screening experiments. CAR usage of HAdV3, 14, 50, and 69 was not studied before, and therefore we experimentally confirmed CAR usage for these HAdV as novel viruses utilizing CAR as a receptor. To rule out that replication of HAdV in transduced CHO cells is responsible for increased transduction rates we performed replication assays on virus genome level, which revealed that there is no HAdV replication. CONCLUSION: In the present study, we screened a HAdV library and identified novel human HAdV using the CAR receptor. To our knowledge, this is the first description of CAR usage for HAdV 3, 14, 50, and 69.

Coxsackievirus and adenovirus receptor mediates the responses of endothelial cells to fluid shear stress.

Endothelial mechanotransduction by fluid shear stress (FSS) modulates endothelial function and vascular pathophysiology through mechanosensors on the cell membrane. The coxsackievirus and adenovirus receptor (CAR) is not only a viral receptor but also a component of tight junctions and plays an important role in tissue homeostasis. Here, we demonstrate the expression, regulatory mechanism, and role of CAR in vascular endothelial cells (ECs) under FSS conditions. Disturbed flow increased, whereas unidirectional laminar shear stress (LSS) decreased, CAR expression in ECs through the Krüppel-like factor 2 (KLF2)/activator protein 1 (AP-1) axis. Deletion of CAR reduced the expression of proinflammatory genes and endothelial inflammation induced by disturbed flow via the suppression of NF-κB activation. Consistently, disturbed flow-induced atherosclerosis was reduced in EC-specific CAR KO mice. CAR was found to be involved in endothelial mechanotransduction through the regulation of platelet endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation. Our results demonstrate that endothelial CAR is regulated by FSS and that this regulated CAR acts as an important modulator of endothelial mechanotransduction by FSS.

The Coxsackievirus and Adenovirus Receptor, a Required Host Factor for Recovirus Infection, Is a Putative Enteric Calicivirus Receptor.

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in both developed and developing countries. Studies of HuNoV host cell interactions are limited by the lack of a simple, robust cell culture system. Due to their diverse HuNoV-like biological features, including histo-blood group antigen (HBGA) binding, rhesus enteric caliciviruses (ReCVs) are viable surrogate models for HuNoVs. In addition, several ReCV strains can be propagated to high titers in standard nonhuman primate cell lines while causing lytic infection and cell death. To identify the ReCV entry receptor, we performed CRISPR/Cas9 library screening in Vero cells, which identified the coxsackievirus and adenovirus receptor (CAR) as a candidate ReCV entry receptor. We showed that short interfering RNA, anti-human CAR (hCAR) monoclonal antibody RmcB treatment, and recombinant hCAR ectodomain blocked ReCV replication in LLC-MK2 cells. CRISPR/Cas9-targeted knockout of CAR in LLC-MK2 and Vero cells made these cell lines resistant to ReCV infection, and susceptibility to infection could be restored by transient expression of CAR. CHO cells do not express CAR or HBGAs and are resistant to ReCV infection. Recombinant CHO cells stably expressing hCAR or the type B HBGA alone did not support ReCV infection. However, CHO cells expressing both hCAR and the type B HBGA were susceptible to ReCV infection. In summary, we have demonstrated that CAR is required for ReCV infection and most likely is a functional ReCV receptor, but HBGAs are also necessary for infection.IMPORTANCE Because of the lack of a simple and robust human norovirus (HuNoV) cell culture system surrogate, caliciviruses still represent valuable research tools for norovirus research. Due to their remarkable biological similarities to HuNoVs, including the utilization of HBGAs as putative attachment receptors, we used rhesus enteric caliciviruses (ReCVs) to study enteric calicivirus host cell interactions. Using CRISPR/Cas9 library screening and functional assays, we identified and validated the coxsackievirus and adenovirus receptor (CAR) as a functional proteinaceous receptor for ReCVs. Our work demonstrated that CAR and HBGAs both are necessary to convert a nonsusceptible cell line to being susceptible to ReCV infection. Follow-up studies to evaluate the involvement of CAR in HuNoV infections are ongoing.

Isoform specific editing of the coxsackievirus and adenovirus receptor.

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.

The Cxadr-Adam10 complex plays pivotal roles in tight junction integrity and early trophoblast development in mice.

Understanding preimplantation embryo development has important implications for assisted reproductive technologies (ARTs) after the introduction of in vitro fertilisation and embryo transfer because most embryonic losses occur during pre/peri-implantation. Recent studies have shown that tight junctions (TJs) are important components for embryos to develop to the blastocyst stage. However, their biological function after cavitation has not been extensively studied. We examined TJ assembly focusing on coxsackievirus and adenovirus receptor (Cxadr) and A disintegrin and metalloproteinase 10 (Adam10) using siRNA and/or an Adam10-specific inhibitor (GI254023X). TJ-associated genes, including occludin and tight junction protein 1 (Tjp1), were downregulated in the Cxadr knockdown (KD) embryos but were unaltered in Adam10 KD embryos. However, Adam10 KD or chemical inhibition affected subcellular localisation of Adam10, Cxadr, and Tjp1, leading to disrupted TJ assembly. Furthermore, Cxadr KD or GI254023X-treated blastocysts showed a relatively smaller outgrowth area and aberrant expression of transcription factor AP-2γ, a trophoblast-specific marker in the in vitro embryo outgrowth assay. In summary, we demonstrated that the Cxadr-Adam10 complex might moderate TJ integrity/stability and play pivotal roles during early embryonic development. Collectively, understanding the establishment of the TJ complex and its integrity will provide insight into translational research for predicting and selecting developmental competency for ART.

Differential expression of coxsackievirus and adenovirus receptor in endomyocardial tissue of patients with myocarditis.

Recent studies demonstrated that the expression of coxsackievirus and adenovirus receptor (CAR) is implicated in the pathophysiology of myocarditis. The aim of the present study was to assess the association between active and borderline myocarditis and CAR expression in endomyocardial tissues, and analyze the association between CAR expression and treatment response. An analytic, cross­sectional, retrospective study was performed in 26 patients with myocarditis and 10 control subjects without heart disease. Myocardial biopsies were obtained and CAR transcription was measured by reverse transcription­quantitative polymerase chain reaction analysis. The association between CAR mRNA levels and the response to immunosuppressive or conventional therapy (treatment responders, n=17; non­responders, n=9) or with the type of histological myocarditis (active myocarditis, n=16; borderline myocarditis, n=10) was analyzed. CAR transcription levels were significantly lower (P=0.012) in patients with myocarditis compared with controls, and a significant decrease was observed (P=0.023) in CAR mRNA levels among patients with borderline myocarditis compared with the no myocarditis group. Patients responding to therapy exhibited higher CAR mRNA levels (P=0.036) compared with patients not responding to treatment, as evaluated based on clinical and echocardiographic criteria (immunosuppressive therapy, n=8; conventional therapy, n=1). Myocarditis in non­responders was associated with fewer clinical manifestations and lower CAR mRNA levels. A significant difference was only found regarding the use of oral steroids in patients with active myocarditis who responded to treatment (P=0.02), with no difference in borderline myocarditis. In conclusion, the transcriptional level of CAR is low in the endomyocardial tissue of patients with myocarditis, and it is lower in borderline myocarditis and in non­responder patients. These findings may enable early identification of patients who may benefit from treatment and timely determination of prognosis.

External validation of molecular subtype classifications of colorectal cancer based on microsatellite instability, CIMP, BRAF and KRAS.

BACKGROUND: Competing molecular classification systems have been proposed to complement the TNM staging system for a better prediction of survival in colorectal cancer (CRC). However, validation studies are so far lacking. The aim of this study was to validate and extend previously published molecular classifications of CRC in a large independent cohort of CRC patients. METHODS: CRC patients were recruited into a population-based cohort study (DACHS). Molecular subtypes were categorized based on three previously published classifications. Cox-proportional hazard models, based on the same set of patients and using the same confounders as reported by the original studies, were used to determine overall, cancer-specific, or relapse-free survival for each subtype. Hazard ratios and confidence intervals, as well as Kaplan-Meier plots were compared to those reported by the original studies. RESULTS: We observed similar patterns of worse survival for the microsatellite stable (MSS)/BRAF-mutated and MSS/KRAS-mutated subtypes in our validation analyses, which were included in two of the validated classifications. Of the two MSI subtypes, one defined by additional presence of CIMP-high and BRAF-mutation and the other by tumors negative for CIMP, BRAF and KRAS-mutations, we could not confirm associations with better prognosis as suggested by one of the classifications. For two of the published classifications, we were able to provide results for additional subgroups not included in the original studies (men, other disease stages, other locations). CONCLUSIONS: External validation of three previously proposed classifications confirmed findings of worse survival for CRC patients with MSS subtypes and BRAF or KRAS mutations. Regarding MSI subtypes, other patient characteristics such as stage of the tumor, may influence the potential survival benefit. Further integration of methylation, genetic, and immunological information is needed to develop and validate a comprehensive classification that will have relevance for use in clinical practice.

A tumor targeting oncolytic adenovirus can improve therapeutic outcomes in chemotherapy resistant metastatic human breast carcinoma.

Breast cancer is the most prevalent malignancy in women, which remains untreatable once metastatic. The treatment of advanced breast cancer is restricted due to chemotherapy resistance. We previously investigated anti-cancer potential of a tumor selective oncolytic adenovirus along with cisplatin in three lung cancer cells; A549, H292, and H661, and found it very efficient. To our surprise, this virotherapy showed remarkable cytotoxicity to chemo-resistant cancer cells. Here, we extended our investigation by using two breast cancer cells and their resistant sublines to further validate CRAd's anti-resistance properties. Results of in vitro and in vivo analyses recapitulated the similar anti-tumor potential of CRAd. Based on the molecular analysis through qPCR and western blotting, we suggest upregulation of coxsackievirus-adenovirus receptor (CAR) as a selective vulnerability of chemotherapy-resistant tumors. CAR knockdown and overexpression experiments established its important involvement in the success of CRAd-induced tumor inhibition. Additionally, through transwell migration assay we demonstrate that CRAd might have anti-metastatic properties. Mechanistic analysis show that CRAd pre-treatment could reverse epithelial to mesenchymal transition in breast cancer cells, which needs further verification. These insights may prove to be a timely opportunity for the application of CRAd in recurrent drug-resistant cancers.

CXADR-Mediated Formation of an AKT Inhibitory Signalosome at Tight Junctions Controls Epithelial-Mesenchymal Plasticity in Breast Cancer.

Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial-mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here, we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyperactivation of AKT and sensitized cells to TGFß1-induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKT-inhibitory signalosome at TJ and regulates epithelial-mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer. SIGNIFICANCE: The tight junction protein CXADR controls epithelial-mesenchymal plasticity in breast cancer by stabilizing the AKT regulators PTEN and PHLPP2.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/47/F1.large.jpg.