Expression Of Coxsackievirus-adenovirus Receptor Research Articles

CAR expression in invasive breast carcinoma and its effect on adenovirus transduction efficiency

BackgroundBreast cancer is the second leading cause of death in women, with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) as the two most common forms of invasive breast cancer. While estrogen receptor positive (ER+) IDC and ILC are treated similarly, the multifocality of ILC presents challenges in detection and treatment, worsening long-term clinical outcomes in patients. With increasing documentation of chemoresistance in ILC, additional treatment options are needed. Oncolytic adenoviral therapy may be a promising option, but cancer cells must express the coxsackievirus & adenovirus receptor (CAR) for adenoviral therapy to be effective. The present study aims to evaluate the extent to which CAR expression is observed in ILC in comparison to IDC, and how the levels of CAR expression correlate with adenovirus transduction efficiency. The effect of liposome encapsulation on transduction efficiency is also assessed.MethodsTo characterize CAR expression in invasive breast carcinoma, 36 formalin-fixed paraffin-embedded (FFPE) human breast tumor samples were assayed by CAR immunohistochemistry (IHC). Localization of CAR in comparison to other junctional proteins was performed using a multiplex immunofluorescence panel consisting of CAR, p120-catenin, and E-cadherin. ILC and IDC primary tumors and cell lines were transduced with E1- and E3-deleted adenovirus type 5 inserted with a GFP transgene (Ad-GFP) and DOTAP liposome encapsulated Ad-GFP (DfAd-GFP) at various multiplicities of infection (MOIs). Transduction efficiency was measured using a fluorescence plate reader. CAR expression in the human primary breast carcinomas and cell lines was also evaluated by IHC.ResultsWe observed membranous CAR, p120-catenin and E-cadherin expression in IDC. In ILC, we observed cytoplasmic expression of CAR and p120-catenin, with absent E-cadherin. Adenovirus effectively transduced high-CAR IDC cell lines, at MOIs as low as 12.5. Ad-GFP showed similar transduction as DfAd-GFP in high-CAR IDC cell lines. Conversely, Ad-GFP transduction of ILC cell lines was observed only at MOIs of 50 and 100. Furthermore, Ad-GFP did not transduce CAR-negative IDC cell lines even at MOIs greater than 100. Liposome encapsulation (DfAd-GFP) improved transduction efficiency 4-fold in ILC and 17-fold in CAR-negative IDC cell lines.ConclusionThe present study demonstrates that oncolytic adenoviral therapy is less effective in ILC than IDC due to differences in spatial CAR expression. Liposome-enhanced delivery may be beneficial for patients with ILC and tumors with low or negative CAR expression to improve adenoviral therapeutic effectiveness.

Treatment of Human Pancreatic Cancers Following Local and Systemic Administration of Oncolytic Adenovirus Serotype 35.

Oncolytic adenoviruses (Ads) (OAds) are gaining attention as an effective remedy for pancreatic cancer. Most OAds are based on human Ad serotype 5 (Ad5) (OAd5); however, two major drawbacks of OAd5 have been reported. Expression of coxsackievirus-adenovirus receptor, a primary infection receptor of Ad5, is often decreased on malignant tumor cells, including pancreatic cancers. More than 60% of adults have neutralizing antibodies against Ad5. Previously, we developed an OAd composed of Ad serotype 35 (Ad35) (OAd35). Ad35 recognizes CD46, which is often up-regulated on pancreatic cancers. In addition, only 20% or fewer adults have anti-Ad35 neutralizing antibodies. We examined the tumor cell lysis activities of OAd35 in the four human pancreatic cancer cell lines in the presence and absence of human serum. The tumor growth suppression effects of OAd35 after local and systemic administration were evaluated in nude mice bearing human pancreatic tumors. OAd35 showed higher levels of tumor cell lysis activities than OAd5 in the human pancreatic cancer cell lines AsPC-1 and BxPC-3. Although the in vitro tumor cell lysis activities of OAd5 against MIA PaCa-2 and PANC-1 cells were strongly attenuated in the presence of human serum, OAd35 mediated comparable levels of tumor cell lysis in the presence and absence of human serum. Systemic administration of OAd5 did not mediate significant growth inhibition against the subcutaneous BxPC-3 tumor. On the other hand, OAd35 significantly suppressed tumor growth. OAd35 would be suitable as an alternative anticancer agent for pancreatic cancer.

Abstract 3551: Oncolytic adenovirus serotype 35-mediated activation of NK cells leads to efficient anti-tumor effects

Abstract Oncolytic adenoviruses (OAds) are getting much attention as cancer immunotherapy agents. Almost all OAds are based on human adenovirus serotype 5 (Ad5) (OAd5). However, OAd5 has several drawbacks which possibly attenuate the anti-tumor effects of OAd5. More than 80% of adults have neutralizing antibodies against Ad5. The expression of coxsackievirus-adenovirus receptor (CAR), which is the primary infection receptor of Ad5, often declines on malignant tumor cells. Previously, we have developed a novel OAd fully composed of human adenovirus serotype 35 (Ad35) (OAd35) (Ono et al., Mol.Ther.Oncolytics, 2021). Only 20% or fewer adults have neutralizing antibodies against Ad35. Ad35 recognizes CD46, which is highly expressed on malignant tumor cells, as an infection receptor. OAd35 showed efficient tumor cell lysis activities in many types of tumor cells; however, the mechanism of anti-tumor effects of OAd35 remains to be fully evaluated. In this study, we examined the mechanism of anti-tumor effects of OAd35. Although OAd35 showed efficient tumor growth suppression at levels comparable to OAd5 following intratumoral administration in nude mice bearing H1299 human lung cancer xenografts, the genome copy numbers of OAd35 in the tumor were about 500-folds lower than those of OAd5 at 18 days after administration and were almost background levels. These data suggest that other mechanisms different from infection of tumor cells with OAd35 were involved in the anti-tumor effects of OAd35. Since previous studies reported that Ad35 efficiently activated innate immunity in natural killer (NK) cells, we examined whether OAd35-mediated activation of NK cells contributed to the anti-tumor effects of OAd35. Intratumoral administration of OAd35 resulted in efficient infiltration and activation of NK cells in H1299 tumors. In contrast, OAd5 did not apparently induce the infiltration and activation of NK cells in the tumors. OAd35-mediated activation and infiltration of NK cells were also found in B16 tumor-bearing immunocompetent mice. Next, in order to examine the roles of NK cells in the anti-tumor effects of OAd35, NK cells were depleted by intravenous injection of anti-GM1 antibody in nude mice, followed by intratumoral administration of OAd35. The depletion of NK cells significantly attenuated the anti-tumor effects of OAd35 on H1299 tumors. Furthermore, pretreatment with anti-GM1 antibody significantly increased the genome copy numbers of OAd35 in the tumor, suggesting that activated NK cells removed the OAd35-infected tumor cells. This study demonstrated that OAd35 significantly activated NK cells following intratumoral administration and that activated NK cells efficiently removed the tumor cells, leading to efficient tumor growth suppression. Citation Format: Ryosuke Ono, Kosuke Takayama, Fuminori Sakurai, Hiroyuki Mizuguchi. Oncolytic adenovirus serotype 35-mediated activation of NK cells leads to efficient anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3551.

Pregnancy Loss Following Coxsackievirus B3 Infection in Mice during Early Gestation Due toHigh Expression of Coxsackievirus-Adenovirus Receptor (CAR) in Uterus and Embryo

Coxsackieviruses are important pathogens in children and the outcomes of neonatal infection can be serious or fatal. However, the outcomes of coxsackievirus infection during early gestation are not well defined. In this study, we examined the possibility of vertical transmission of coxsackievirus B3 (CVB3) and the effects of CVB3 infection on early pregnancy of ICR mice. We found that the coxsackievirus and adenovirus receptor (CAR) was highly expressed not only in embryos but also in the uterus of ICR mice. CVB3 replicated in the uterus 1 to 7 days post-infection (dpi), with the highest titer at 3 dpi. The pregnancy loss rate in mice infected with CVB3 during early gestation was 38.3%, compared to 4.7% and 2.7% in mock-infected and UV-inactivated-CVB3 infected pregnant mice, respectively. These data suggest that the uterus and embryo, which express abundant CAR, are important targets of CVB3 and that the vertical transmission of CVB3 during early gestation induces pregnancy loss.

Enhanced Antitumor Efficacy of Integrin-targeted Oncolytic Adenovirus AxdAdB3-F/RGD on Bladder Cancer

To evaluate the therapeutic efficacy of AxdAdB-3 with Arg-Gly-Asp (RGD)-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection in bladder cancers. Flow cytometric analysis was applied to evaluated adenovirus-mediated gene transduction into various cells. The cytopathic effects of AxdAdB3-F/RGD were evaluated in bladder cancer cell lines and a normal bladder mucosa-derived cell line (HCV29) with AxCAZ3-F/RGD (control) or AxdAdB-3. The efficacy of bladder instillation therapy with AxdAdB3-F/RGD for orthotopic bladder cancer was investigated in nude mice. Expression of coxsackievirus adenovirus receptor (CAR) and integrins (αvβ3 and αvβ5) vary in different bladder cancer cell lines. The susceptibility of various cell lines to adenovirus was associated with the expression of CAR. AxdAdB-3 was more cytopathic in CAR-positive bladder cancer cells than in CAR-negative cells, whereas AxdAdB3-F/RGD caused effective oncolysis in both CAR-positive and CAR-negative bladder cancer cells. AxdAdB3-F/RGD was not cytotoxic to HCV29 cells. Direct instillation of AxdAdB3-F/RGD into the bladder of the orthotopic model, established by CAR-deficient human bladder cancer cells, inhibited tumor growth and led to significantly elongated survival. E1A and E1B double-restricted oncolytic adenovirus with RGD fiber modification has enhanced infectivity and oncolytic effects to CAR-deficient bladder cancers, suggesting the therapeutic potential of AxdAdB3-F/RGD for bladder cancers.

Hepatic damage caused by coxsackievirus B3 is dependent on age-related tissue tropisms associated with the coxsackievirus-adenovirus receptor

Coxsackievirus B (CVB) and enterovirus 71 (EV71) are important causes of severe enteroviral diseases in neonates or young children in Taiwan. CVB can cause fulminant hepatitis, myocarditis or meningoencephalitis. This study was designed to explore the role of coxsackievirus-adenovirus receptor (CAR) in the pathogenesis of CVB3-infected hepatocytes via in vitro and mice studies. CVB3 (CVB3/2630) was isolated from liver tissue of a neonate with fulminant hepatitis. Cell lines A549, HeLa, HEp2 and Huh-7 were maintained in Dulbecco's modified Eagle's medium. Mice progeny 1 or 7 days old were used in the experiments. Viremia was noted in 7-day-old ICR mice 2 h after intraperitoneal injection. The highest viral titers were detected in blood, liver and spleen. Histopathological studies of the liver demonstrated polymorphonuclear cell infiltration, massive hepatic cell necrosis and apoptosis. CAR was expressed more in liver than in other tissues. Expression of CAR decreased with mouse age. Anti-CAR monoclonal antibody prevented infection of Huh-7 cells from CVB3. Furthermore, anti-CAR monoclonal antibody pretreatment can reduce mortality and decrease the level of liver enzymes in CVB3-infected mice. These findings indicate that CAR plays an important role in the initiation of CVB infections and is closely associated with hepatotropism and age-specific susceptibility.

Inhibition of Retinoblastoma In Vitro and In Vivo with Conditionally Replicating Oncolytic Adenovirus H101

To determine the therapeutic effect of oncolytic adenovirus H101 on retinoblastoma in vitro and in vivo. The expression of coxsackievirus-adenovirus receptor (CAR) in human retinoblastoma cell line HXO-RB(44) was determined by RT-PCR, Western blot, immunofluorescence, and immunocytochemistry staining. Appropriate multiplicity of infection was determined using flow cytometry in retinoblastoma cells with green fluorescent protein-expressing adenovirus (AdGFP). The viability of HXO-RB(44) cells treated with H101 or AdGFP was measured using a cell counting kit-8-based procedure. Viral proliferation in vitro was measured by end point dilution titration and real-time PCR. Cell cycle and apoptotic activity of HXO-RB(44) were analyzed by flow cytometry. NOD-SCID mice bearing retinoblastoma xenografts were treated with intratumoral injection of H101, AdGFP, or PBS. Tumor volume and survival time were recorded. Immunohistochemistry for adenoviral fiber protein and Western blot for adenoviral Hexon protein of retinoblastoma xenografts were performed to evaluate H101 virus replication in vivo. HXO-RB(44) cells expressed CAR and were sensitive to adenoviral infection. HXO-RB(44) cells treated with H101 had reduced cell viability compared with AdGFP-treated cells (P < 0.01). Abundant replication of H101 in HXO-RB(44) cells resulted in G(2)/M-phase arrest and finally tumor cell lysis, but the apoptosis pathway was not activated. Tumor-bearing mice treated with H101 had reduced tumor burdens and prolonged survival times compared with PBS and AdGFP controls (both P < 0.01). Immunohistochemical and Western blot examination revealed widespread replication of H101 within the tumor. These results suggest that H101 effectively inhibits the growth of retinoblastoma cells in vitro and in mice and may serve as a novel therapy for retinoblastoma.

Activation of STAT1 transcription factor precedes up-regulation of coxsackievirus–adenovirus receptor during viral myocarditis

The coxsackievirus-adenovirus receptor (CAR) was originally described as a transmembrane protein involved in viral infection and was later found to be required for normal heart development. However, the role of CAR in virus-induced myocarditis has not been investigated so far. The purpose of this study was to assess myocardial CAR expression in response to cytokine-induced inflammatory reactions during the course of coxsackievirus-induced myocarditis. In Balb/c mice intraperitoneally infected with either 2x10(4) plaque-forming units (PFUs) of coxsackie B3 virus (CVB3) or 10(2) PFUs CVB3, CAR expression and tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1), a known cytokine-inducible transcription factor involved in viral defense, were determined. Our results demonstrated that within the first 7 days after virus inoculation, when the viral replication and STAT1 activation in the heart tissue was most prominent, the expression of CAR did not surpass that of uninfected controls. However, the up-regulation of CAR was observed 9 weeks later, when enteroviral RNA was no longer detectable and activation of STAT1 had already ceased. In contrast to the STAT1 target genes Mig and Irf1, interferon gamma stimulation failed to up-regulate Car expression in isolated cardiomyocytes. In human endomyocardial biopsies, Car expression was found to be elevated in approximately one third of patients with dilated cardiomyopathy (9 of 30 patients) as compared with controls. Thus, activation of STAT1 clearly precedes the enhanced CAR expression observed during tissue reorganization, suggesting an essential role of STAT1 transcription factors in orchestrating the sequential actions involved in adaptive immune response.

E1A, E1B Double-Restricted Adenovirus with RGD-Fiber Modification Exhibits Enhanced Oncolysis for CAR–Deficient Biliary Cancers

Cancers of biliary system represent highly malignant diseases of dismal prognosis. We have previously introduced AxdAdB3, an E1A, E1B double-restricted oncolytic adenovirus, which showed excellent oncolytic efficacy for approximately half of the biliary cancer lines with an enhanced safety to normal cells. The purpose of this study was to evaluate whether RGD-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection, can improve the infectivity and efficacy of AxdAdB3 for biliary cancers. Expressions of adenoviral receptors, coxsackievirus adenovirus receptor (CAR) and integrins (alpha(v)beta(3) and alpha(v)beta(5)), were compared with the level of infectivity of LacZ-expressing replication-defective adenoviruses with wild-type fibers or RGD-modified fibers in a panel of biliary cancer cell lines in vitro. Viral replication and cytotoxicity in vitro of AxdAdB3-F/RGD, a novel E1A, E1B double-restricted replication-selective adenovirus with RGD-modified fibers, were compared with those of its parent virus, AxdAdB3, in various biliary cancer cells and in normal cells. In vivo antitumor effects of these oncolytic viruses were compared in a xenograft tumor model. Expression of CAR significantly correlated with the adenovirus infectivity, whereas integrin alpha(v)beta(5) was abundantly expressed in almost all biliary cancer cells. Whereas AxdAdB3 effectively replicated and lysed only the biliary cancer cells with a preserved expression of CAR, AxdAdB3-F/RGD exhibited efficient replication and potent oncolysis in both CAR-positive and CAR-negative biliary cancer cells. AxdAdB3-F/RGD showed attenuated replication and little cytopathy in human normal cells (i.e., hepatocytes, WI-38 cells) as well as AxdAdB3. Furthermore, in nude mice with s.c. xenografts of CAR-deficient human biliary cancer, i.t. AxdAdB3-F/RGD therapy caused a marked inhibition of tumor growth. The RGD-fiber modification strategy enhanced the infectivity, replication, and oncolytic effects of the E1A, E1B double-restricted oncolytic adenovirus for CAR-deficient biliary cancers. In addition, it preserved the merit of excellent safety of the double-restricted virus for normal cells. These results suggest a potential use of this agent for the treatment of biliary cancers.

Coxsackievirus and adenovirus receptor expression in non-malignant lung tissues and clinical lung cancers

Adenoviral vector mediated gene delivery has been applied in clinical trials and mechanistic studies to explore new treatment approaches for lung cancers. The expression of coxsackievirus adenovirus receptor (CAR), the primary receptor for the most commonly used adenovirus serotype 5 (Ad5)-based vectors, predominantly determines the permissiveness of lung cancer cells. CAR expression is also suggested to modulate tumor cell proliferation capacity. Here, we studied CAR expression in archival lung cancer specimens by using well-characterized CAR 72 antibodies. High levels of CAR expression were observed in most of the 32 cases of squamous cell carcinoma lung cancers and in all the five cases of small cell lung cancers investigated. In contrast, high levels of CAR expression were detected only in 6 of 22 adenocarcinoma lung cancers. The relative levels of CAR expression did not correlate with the pathologic grade in lung cancers, and was thus inconsistent with a role of modulating cancer cell proliferation. Of note, CAR expression was not detected in non-malignant alveolar cells. Our data suggest a preferred utility of Ad5 vector mediated gene delivery to squamous cell carcinoma lung cancers, small cell lung cancers, but not to the majority of adenocarcinoma lung cancers.

Effective gene transfer to human melanomas via integrin-targeted adenoviral vectors.

The utility of recombinant adenoviral vectors (Adv) for gene therapy is limited by their low transduction efficiency and lack of specificity for target cells. The low transduction efficiency is often recognized as due to deficiency of the primary adenoviral receptor, the coxsackievirus-adenovirus receptor (CAR). In this paper, studies of CAR levels on human melanoma cell lines confirmed that low transduction efficiency was closely related to deficiency of the adenoviral receptor. To achieve CAR-independent gene transfer via Adv, we modified viral tropism via genetic alteration of the adenovirus type 5 (Ad5) fiber protein. Insertion of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain allowed the virus to use an alternative receptor, the integrin receptor, during the cell entry process. With this modified vector (Adv-F/RGD) transduction was increased 5- to 96-fold relative to a vector containing wild-type fiber (Adv-F/wt) in five human melanoma cells expressing integrins of the alpha(v)beta(3), alpha(v)beta(5) class, which are recognized by the RGD peptide motif. In contrast, no significant difference in transduction efficiency between Adv-F/RGD and Adv-F/wt was observed in 293 cells, which show high-level expression of CAR. In this study, we attempted to apply Adv-F/RGD for gene therapy for malignant melanoma. At the same multiplicity of infection, melanoma cells infected with Adv-F/RGD carrying human interleukin 2 (AxCAhIL2-F/RGD) produced a higher level of cytokine than cells infected with AxCAhIL2-F/wt. Treatment by intratumoral injection of AxCAhIL2-F/RGD was more effective than intratumoral injection of AxCAhIL2-F/wt in regressing tumors in a melanoma xenograft model. These data suggest that integrin-targeted adenoviral vectors may be a powerful tool in gene therapy for CAR-deficient melanomas.

Adenovirus vector-induced inflammation: capsid-dependent induction of the C-C chemokine RANTES requires NF-kappa B.

Adenovirus vectors for gene therapy activate responses in the host that result in acute inflammation of transduced tissues. Our previous studies in vivo demonstrate that chemokines, including the C-C chemokine RANTES (regulated on activation, normal T cell expressed and secreted), contribute to the acute inflammation induced by adenovirus vectors. Various first-generation adenovirus vectors, including adCMV beta gal, were equally capable of inducing the expression of RANTES 3 hr after transduction in epithelial HeLa and REC cells. Deletional analysis of the human RANTES promoter revealed that induction by adCMV beta gal required the elements spanning base pairs -90 to -25 of the gene. Electrophoretic mobility shift assays demonstrated that nuclear extracts from adCMV beta gal-transduced HeLa cells bound to an NF-kappa B site at position -54. Overexpression of I-kappa B alpha suppressed adCMV beta gal induction of RANTES, confirming that this process was dependent on the nuclear translocation of NF-kappa B. The coxsackievirus-adenovirus receptor (CAR)-independent, serotype 3 adenovirus was equally capable of inducing the expression of RANTES in HeLa cells. This observation suggested that binding to CAR was not specifically required in adenovirus vector-induced RANTES expression. The use of RGD peptides to block adCMV beta gal interactions with alpha(v)-integrins reduced RANTES expression but also transduction efficiency. In CAR-deficient P815 cells, binding of adCMV beta gal to alpha(v)-integrins without efficient cell transduction did not result in increased RANTES expression. Expression of human CAR in P815 cells increased the binding and transduction efficiency of adCMV beta gal and resulted in RANTES expression in these cells. These results suggest that the induction of RANTES by adenovirus vectors is dependent on efficient interaction with its cell surface receptors and vector internalization. Understanding the biology of the host response to adenovirus vectors will impact the design of future generations of these agents aimed at reducing their immunogenicity and improving their safety.