The adenovirus (Ad) serotype 5 genome encodes two noncoding small RNAs (virus-associated RNAs I and II [VA-RNAI and -II]), which are approximately 160-nucleotide (nt) RNAs transcribed by RNA polymerase III. It is well known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI and -II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and mivaRNAII in Ad replication have remained to be clarified. In this study, we demonstrated mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant forms of mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin 4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via posttranscriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated downregulation of CUL4A enhanced JNK signaling and thereby promoted Ad infection. IMPORTANCE Several types of viruses encode viral miRNAs which regulate host and/or viral gene expression via posttranscriptional gene silencing, leading to efficient viral infection. Adenovirus (Ad) expresses miRNAs derived from V-RNAs (mivaRNAI and -II); however, recent studies have revealed that processing of VA-RNAI into mivaRNAI inhibits Ad replication. Conversely, we demonstrate here that mivaRNAII significantly promotes Ad replication and that mivaRNAII-mediated suppression of CUL4A expression via posttranscriptional gene silencing induces accumulation of c-Jun, leading to promotion of Ad infection. These results exhibited the significance of VA-RNAII for supporting Ad infection through a mechanism complementary to that of VA-RNAI. These observations could provide important clues toward a new perspective on hos-virus interaction. Moreover, Ad is widely used as a basic framework for viral vectors and oncolytic viruses. Our findings will help to regulate Ad infection and will promote the development of novel Ad vectors and oncolytic Ad.

Anaplastic lymphoma kinase (ALK)-rearrangements are mainly encountered in 5% of adenocarcinomas lung cancer (Ad-LC) patients and anti-ALK targeted therapy dramatically improves therapeutic responses. The prevalence of ALK rearrangement in squamous cell lung carcinomas (Sq-LC) is extremely rare and thus, clinicopathological features and clinical outcomes for ALK inhibitors of ALK-rearranged Sq-LC were still unknown. Accordingly, in this study, we compared clinical features and clinical outcomes in patients with Sq-LC and Ad-LC. We retrospectively analyzed the clinical features of five patients with ALK-rearranged Sq-LCs including two ALK-rearranged adenosquamous cell lung carcinomas (AdSq-LC) and compared the results with ALK-rearranged Ad-LC. We also evaluated representative cases of both responder and nonresponder to ALK inhibitors. The prevalence of ALK rearrangement in Sq-LCs was 1.36%. The population in ALK rearrangement NSCLC with smoking history was higher in ALK-rearranged Sq-LC than in ALK-rearranged Ad-LCs (80.0% and 68.0%). Progression-free survival (PFS) after initial treatment with the ALK inhibitor crizotinib was significantly shorter in ALK-rearranged Sq-LC than in ALK-rearranged Ad-LC (6.4±4.7 months and 13.4±12.8 months: p=0.033). Notably, two ALK fluorescence in situ hybridization (FISH)-positive/immunohistochemistry-negative cases did not respond to crizotinb, and PFS following alectinib treatment of ALK-rearranged Sq-LC was short (p = 0.045). The responder to ceritinib showed the presence of the L1196M mutation, which causes other ALK inhibitor resistance, by rebiopsy and successes to maintain CR response, even if detected off-target resistance marker of both EGFR and vimentin, a marker of EMT, for ALK inhibitors. However, the nonresponder did not respond to all ALK inhibitors, despite the presence of ALK FISH-positive circulating tumor cells and circulating free DNA without the mutation for ALK inhibitors resistance by liquid-biopsy. ALK-rearranged Sq-LC was associated with poor outcomes in ALK inhibitor-treated patients, suggesting that complexity of resistance mechanisms including off-target mechanisms for ALK inhibitors may exist. Oncologists should be aware of the possibility of ALK-rearranged Sq-LC based on clinicopathological features and plan the next therapeutic strategy by as much of re-biopsy accordingly to improve clinical outcomes.

The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α significantly enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB leads to suppression of Ad vector-mediated tissue damages via not only suppression of inflammatory responses but also reduction in the leaky expression of Ad genes.

Reovirus has gained much attention as an anticancer agent; however, the mechanism of the tumor cell-specific replication of reovirus is not fully understood. Although Ras activation is known to be crucial for tumor cell-specific replication of reovirus, it remains controversial which cellular factors are required for the reovirus-mediated tumor cell killing. In this study, we systematically investigated which cellular factors determined the efficiencies of reovirus-mediated tumor cell killing in various human cultured cell lines. The efficiency of reovirus-mediated cell killing varied widely among the cell lines. Junction adhesion molecule-A, a reovirus receptor, was highly expressed in almost all cell lines examined. Ras activation levels were largely different between the cell lines; however, there were no apparent correlations among the reovirus-mediated cell killing efficiencies and Ras activation status. On the other hand, activity levels of the cysteine proteases cathepsins B and L, which are crucial for proteolytic disassembly of the outer capsid proteins of reovirus, showed a tendency to be correlated with the efficiency of reovirus-mediated cell killing. These results indicate that the activity of cathepsins B and L is the most suitable as a biomarker for the efficacy of reovirus-mediated oncolysis among the factors examined in this study.

Altered N-glycosylation of membrane proteins is associated with malignant transformation of cells. We found that the expression of the β4-galactosyltransferase 2 (β4GalT2) gene is decreased markedly during the transformation. Here, we examined whether the tumor growth activity of B16-F10 mouse melanoma cells can be reduced by the enhanced expression of the β4GalT2 gene. We isolated a clone, B16-β4GalT2, showing its β4GalT2 transcript 2.5 times higher than a control clone, B16-mock, by transducing its cDNA, and transplanted them subcutaneously into C57BL/6 mice to examine their tumor growth activity. The results showed that the average size of tumors formed with B16-mock cells is 13.1±0.76 mm, whereas that of tumors formed with B16-β4GalT2 cells is 5.1±1.13 mm (P<0.01) 2 weeks after transplantation. Immunohistochemical analyses showed that the apoptosis and the suppression of angiogenesis are induced in the tumors upon transduction of the β4GalT2 gene. To pursue a clinical usefulness of the β4GalT2 gene for suppressing human tumor growth, we injected adenoviruses carrying the human β4GalT2 cDNA into HuH-7 human hepatocellular carcinomas developed in severe combined immunodeficient mice, and observed marked growth retardation of the tumors. The enhancement of the β4GalT2 gene expression in tumors is one of the promising approaches to suppress human tumor growth.

Recent studies have demonstrated that small double-stranded RNAs (dsRNAs) complementary to the promoter region of target genes enhance the expression of those genes following transfection into cells. Here we show that expression of the matrix metalloproteinase (MMP) inhibitor RECK is activated in the cultured tumor cell lines by transfection with dsRNA complementary to the promoter of the RECK gene, leading to suppression of the expression of MMPs and it inhibited tumor cell invasion. These results support the suggestion that dsRNA complementary to the promoter region of tumor suppressor genes would have potential as a novel antitumor agent.

Background: Cell-based therapy using pancreatic islet cells has emerged as a promising new approach for the treatment of diabetes mellitus. Along with the conventional usage of the purified islets for transplantation, our group has recently established a novel dissociated islet cell-based approach, named islet cell sheet transplantation (Transplantation 30: 5943, 2011). In this approach, individually dissociated islet cells are cultured on a specially-defined culture surface to form a thin and contiguous monolayered islet cell sheet. Since it is known that islet cells are susceptible to apoptotic process during the dispersed cell status, the present study conduced a gene transduction of an anti-apoptotic gene FNK, during the culture period for achieving higher cell survival as well as higher functionality. Methods: Fiber knob-modified adenoviral vectors expressing FNK or GFP under the control of CAG promoter, Ad-CA-K7-GFP, Ad-CA-K7-FNK, were prepared. Purified rat islet cells were dissociated into single islet cells, followed by their culturing on laminin-5-coated and temperature-responsive polymer-immobilized plastic dishes. Cells were incubated with the Ad-vectors at day 1 for 1 hr at a previously defined vector dose, MOI=10. Apoptosis was then induced by inoculating hydrogen peroxide into culture medium at 400μM. Results: After infection with Ad-CA-K7-GFP, approximately 90% of the dispersed-cultured islet cells were found to be gene-transduced. There were no Ad-vector infection-mediated cellular damages. In an another set of experiment, cells were infected with Ad-CA-K7-FNK, and hydrogen peroxide were inoculated on day 2 for the induction of apoptosis. FNK-transduced cultured islet cells showed significantly less caspase expressions as well as remarkable resistance to the apoptosis. Finally, the FNK-transduced islet cells could be harvested as an intact cell sheet format that was expected to produce higher levels of insulin. Conclusions:The present study described a new proof-of-concept approach to engineer apoptosis-resistant and highly functional islet cell construct for transplantation.

Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (AdΔVR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and AdΔVR-shRNA are a powerful framework for shRNA-mediated knockdown.

Carrier cells delivering a conditionally replicating adenovirus (CRAd), which selectively replicates in tumor cells and induces tumor cell lysis, have promising potential for treatment of cancer because CRAd-loaded carrier cells evade inhibition by neutralizing anti-adenovirus (Ad) antibodies and because the carrier cells are locally retained at the injection point after local injection. A previous study by Hamada et al. demonstrated that carrier cells (CRAd-containing cell fragments derived from the carrier cells) are engulfed into the target cells, probably through a pathway independent of the primary receptor for Ad, the coxsackievirus and Ad receptor (CAR) (Mol Ther, 15: 1121–1128; 2007); however, it remains to be elucidated whether carrier cells infected with a conventional CRAd, which is composed of subgroup-C Ad serotype-5 (Ad5), mediate antitumor effects on CAR-negative cells. In order to examine whether carrier cells delivering a conventional CRAd (Carrier-F5) induce lysis of CAR-negative tumor cells, CAR-positive and CAR-negative tumor cells were incubated with Carrier-F5. Carrier-F5 mediated efficient killing of CAR-positive tumor cells; however, CAR-negative tumor cells were almost refractory to Carrier-F5. On the other hand, carrier cells loaded with a fiber-substituted CRAd containing fiber proteins of Ad serotype-35 (Ad35) (CRAd-F35), which binds to human CD46 for infection, showed efficient killing of both CAR-positive and CAR-negative tumor cells. Intra-tumoral injection of carrier cells loaded with CRAd-F35 (Carrier-F35) also resulted in efficient regression of both CAR-positive and CAR-negative tumors. These results demonstrated that the expression levels of receptors for Ad are an important factor for CRAd-loaded carrier cell-mediated cancer therapy, and that Carrier-F35 would have potential as a cancer treatment for not only CAR-positive tumors but also CAR-negative tumors.

Cancer gene therapy by adenovirus vectors (Advs) for metastatic cancer is limited because systemic administration of Adv produces low therapeutic effect and severe side effects. In this study, we generated a dual cancer-specific targeting vector system by using PEGylation and the telomere reverse transcriptase (TERT) promoter and attempted to treat experimental metastases through systemic administration of the vectors. We first optimized the molecular size of PEG and modification ratios used to create PEG-Ads. Systemic administration of PEG-Ad with 20-kDa PEG at a 45% modification ratio (PEG[20K/45%]-Ad) resulted in higher tumor-selective transgene expression than unmodified Adv. Next, we examined the effectiveness against metastases and side effects of a TERT promoter-driven PEG[20K/45%]-Ad containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk). Systemic administration of PEG-Ad-TERT/HSVtk showed superior antitumor effects against metastases with negligible side effects. A cytomegalovirus (CMV) promoter-driven PEG[20K/45%]-Ad also produced antimetastatic effects, but these were accompanied by side effects. Combining PEG-Ad-TERT/HSVtk with etoposide or 5-fluorouracil enhanced the therapeutic effects with negligible side effects. These results suggest that modification with 20-kDa PEG at a 45% modification ratio is the optimal condition for PEGylation of Adv, and PEG-Ad-TERT/HSVtk is a prototype Adv for systemic cancer gene therapy against metastases.

Fiber-substituted adenovirus (Ad) vectors containing fibers of Ad serotype 35 (AdF35) efficiently transduce a variety of human cells because their receptor, human CD46, is ubiquitously expressed on almost all nucleated cells. However, the ubiquitous expression of CD46 might lead to unexpected transduction in untargeted organs. In this study, we developed fiber-modified AdF35 vectors with an integrin-binding Arg-Gly-Asn (RGD) peptide incorporated into the FG, HI or IJ loop, which have been identified as important regions for binding to CD46. Incorporation of foreign peptides into these loops does not inhibit trimerization of the fibers. In CD46-negative cells, fiber-mutant AdF35 vectors containing an RGD peptide in the FG or HI loop showed 6- to 30-fold higher transduction efficiencies in an RGD-peptide-dependent manner than the unmodified AdF35 vectors. In contrast, in CD46-positive cells, insertion of foreign peptides markedly reduced the transduction efficiencies of the AdF35 vectors, indicating that insertion of foreign peptides significantly inhibits binding to CD46. In particular, CD46-mediated transduction was completely diminished by insertion of foreign peptides into the HI loop. Our findings indicate that HI loop is the most suitable domain to mediate a foreign peptide-dependent and CD46-independent transduction by incorporation of foreign peptides into the Ad35 fiber knob.

Dendritic cells (DCs) have a critical role in the induction of antigen-specific immune responses, transporting antigens from peripheral tissue to regional lymph nodes where they interact with antigen-specific T lymphocytes. Recent studies revealed that the efficacy of the T cell-dependent immune response depends on the lifespan of the antigen-presenting DCs in the lymph nodes. Here, we succeeded in engineering long-lived antigen-presenting DCs via Bcl-x L -derived hyperactive mutant antiapoptotic protein (Bcl-x FNK ) gene transfer. In a B16BL6 melanoma model, these long-lived DCs exerted potent antitumor immunity that depended mainly on antigen-specific cytotoxic T lymphocytes. Furthermore, in vivo longevity of the long-lived DC vaccine led to antigen-specific activation of interferon-γ-producing CD4 + and CD8 + T cells. Thus, the long-lived DC vaccine strategy is highly useful for constructing DC vaccines, as well as other cell-based medicines, such as stem cell therapy.