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A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.

A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

Background The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. Materials and methods We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. Results hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. Conclusion Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.

Pemetrexed (PEM) inhibits DNA and RNA synthesis and is currently one of the first‐line agents for mesothelioma. PEM suppresses the activities of several enzymes involved in purine and pyrimidine synthesis, and elevated activity of these enzymes in tumors is often linked with resistance to PEM. The agent also stimulates AMP‐activated protein kinase (AMPK) and consequently influences the mammalian target of rapamycin complex 1 (mTORC1) pathways. Nevertheless, it remains unclear whether PEM resistance is linked to the AMPK or mTORC1 pathways. Here, we established two independent PEM‐resistant mesothelioma cell lines in which expression of the PEM‐target enzymes was not elevated, and found that levels of phosphorylated AMPK and p70S6K and, to a lesser extent, levels of phosphorylated AKT and p53, were increased in these cells as compared with the respective parent cells. PEM stimulation also augmented phosphorylation of AMPK, p70S6K, AKT and p53 in most cases. An AMPK activator increased phosphorylation and PEM resistance in parental cells, and the inhibitor decreased the resistance of PEM‐resistant cells. In contrast, inhibitors for p70S6K and AKT did not influence PEM resistance; furthermore, increased levels of endogenous p53 did not affect PEM sensitivity. These data collectively indicate that constitutive activation of AMPK is associated with PEM resistance, and that this is unconnected with elevated DNA and RNA synthesis.

The differential diagnosis of reactive mesothelial hyperplasia and mesothelioma is difficult. We present a rare case of diffuse pleural thickening with thoracic contraction that was indistinguishable from mesothelioma. A 66-year-old woman with no history of asbestos exposure visited our hospital with a complaint of dyspnea. The clinical findings included circumferential pleural thickening on chest computed tomography image and a high concentration of hyaluronic acid in the pleural fluid. Pleural biopsies obtained by thoracoscopy under local anesthesia were pathologically consistent with mesothelioma, but the patient refused to take any kind of mesothelioma treatments. Four months later, she consented to a surgical pleural biopsy under general anesthesia to obtain larger tissue samples, which included typical proliferating polygonal cells positive for CAM5.2, calretinin, WT-1, D2-40, CK5/6, epithelial membrane antigen, and glucose transporter-1 and negative for carcinoembryonic antigen, BerEP4, and MOC31. The analysis was consistent with diagnosis of epithelioid mesothelioma. Fluorescence in situ hybridization, however, showed the presence of p16 gene, and the expression of BRCA1-associated protein-1 was detected by immunohistochemistry. Our final diagnosis was diffuse pleural thickening unrelated to asbestos exposure. Differential diagnosis of diffuse pleural thickening and malignant mesothelioma is thus difficult and routine immunohistochemical examinations are often insufficient for accurate diagnosis. Multiple diagnostic methods are required for correct diagnosis in a clinically marginal case.

Heat shock protein 90 (HSP90) inhibitors suppressed MDM4 functions which mediated p53 ubiquitination, and blocked a chaperon function which influenced expression of the client proteins. We examined cytotoxic effects of the inhibitors, 17-allylamino-17-demetheoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), on mesothelioma and investigated combinatory effects of the inhibitors and adenoviruses expressing the wild-type p53 gene (Ad-p53). A majority of mesothelioma lacks p14 and p16 expression, which leads to defective p53 pathway despite bearing the wild-type p53 genotype. The HSP90 inhibitors up-regulated endogenous wild-type p53 expression and induced cell death. Furthermore, the inhibitors increased the endogenous p53 levels that were induced by cisplatin. Nevertheless, the HSP90 inhibitors suppressed Ad-p53-induced exogenous p53 expression primarily at a posttranscriptional level and inhibited the Ad-p53-mediated cell death. HSP90 inhibitors suppressed ubiquitination processes which were involved in p53 degradation, but a proteasome inhibitor, MG-132, prevented the HSP90 inhibitors-induced p53 down-regulation. In contrast, an inhibitor for HSP70 with a chaperon function, pifithrin-μ, did not produce the p53 down-regulation. The HSP90 inhibitors did not suppress expression of Ad receptor molecules but rather increased expression of green fluorescence protein transduced by the same Ad vector. These data collectively indicated that an HSP90 inhibitor possessed a divalent action on p53 expression, as an activator for endogenous wild-type p53 through inhibited ubiquitination and a negative regulator of exogenously over-expressed p53 through the proteasome pathway.

Background Pemetrexed (PEM) is an anti-cancer agent targeting DNA and RNA synthesis, and clinically in use for mesothelioma and non-small cell lung carcinoma. A mechanism of resistance to PEM is associated with elevated activities of several enzymes involved in nucleic acid metabolism. Methods We established two kinds of PEM-resistant mesothelioma cells which did not show any increase of the relevant enzyme activities. We screened genes enhanced in the PEM-resistant cells with a microarray analysis and confirmed the expression levels with Western blot analysis. A possible involvement of the candidates in the PEM-resistance was examined with a WST assay after knocking down the expression with si-RNA. We also analyzed a mechanism of the up-regulated expression with agents influencing AMP-activated protein kinase (AMPK) and p53. Results We found that expression of cardiac ankyrin repeat protein (CARP) was elevated in the PEM-resistant cells with a microarray and Western blot analysis. Down-regulation of CARP expression with si-RNA did not however influence the PEM resistance. Parent and PEM-resistant cells treated with PEM increased expression of CARP, AMPK, p53 and histone H2AX. The CARP up-regulation was however irrelevant to the p53 genotypes and not induced by an AMPK activator. Augmented p53 levels with nutlin-3a, an inhibitor for p53 degradation, and DNA damages were not always associated with the enhanced CARP expression. Conclusions These data collectively suggest that up-regulated CARP expression is a potential marker for development of PEM-resistance in mesothelioma and that the PEM-mediated enhanced expression is not directly linked with immediate cellular responses to PEM. Electronic supplementary material The online version of this article (10.1186/s12935-017-0493-8) contains supplementary material, which is available to authorized users.

Background Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. Methods We used type 5 Ad in which the expression of E1A gene was activated by 5′-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. Results The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. Conclusions Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection. Electronic supplementary material The online version of this article (10.1186/s12985-017-0888-0) contains supplementary material, which is available to authorized users.

Although oncolytic adenoviruses are promising cancer therapy agents, for effective oncolytic activity, viruses need to specifically infect and effectively replicate in cancer cells but not in normal cells. We have previously identified a pancreatic cancer‐targeting ligand, SYENFSA (SYE), by screening an adenovirus library displaying random peptides against human pancreatic cancer cells and reported that a survivin promoter‐regulated adenovirus, displaying the SYE ligand (AdSur‐SYE), provided effective oncolysis of pancreatic ductal adenocarcinoma (PDAC) in a preclinical study. As we examined the infectivity of AdSur‐SYE in human surgical specimens of various pancreatic tumors, we unexpectedly found that AdSur‐SYE showed high gene transduction efficiency for pancreatic neuroendocrine tumors (PNETs) as well as for PDAC, 9.1‐ and 6.2‐fold, respectively, compared to that of the nontargeting virus (AdSur). The infectivity of both vectors was almost the same in other cancers and organs such as the pancreas. Immunostaining indicated that the cells infected with AdSur‐SYE were PNET cells but not stromal cells. AdSur‐SYE showed a significantly higher oncolytic potency than that of AdSur in human PNET cell lines, and intratumoral infection with AdSur‐SYE completely diminished subcutaneous tumors in a murine model, in which AdSur‐SYE effectively proliferated and spread. AdSur‐SYE exerted a stronger oncolytic effect in primary PNET cells cocultured with mouse embryonic fibroblasts than AdSur did. Thus, AdSur‐SYE shows promise as a next‐generation therapy for PNET.

Background Replication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting. Methods We constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5′ regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype. Results We found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype. Conclusions Sensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression. Electronic supplementary material The online version of this article (10.1186/s12885-017-3621-x) contains supplementary material, which is available to authorized users.

Original Article, Background: Conventional serum markers frequently yield negative results in the early stages of hepatocellular carcinoma (HCC). Serum IgG autoantibodies to tumor-associated antigens have been reported even in the early stages. However, very little information is available for Japanese patients with HCC. This study aimed to profile serum autoantibodies in Japanese patients with HCC, using enzyme-linked immunosorbent assay (ELISA) systems based on tumor-associated antigens (TAAs) /antigenic fragments. Methods: Sera were obtained from patients with HCC at stages I (n = 11), II (n = 30), and III (n = 9). In total, 18 serum autoantibodies were detected using ELISA. The TAAs used were RalA, Hsp70, p90, KM-HN-1, Ny-ESO-1, Galectin-1, Sui1, p53, Annexin A2, Prx6, VEGF, c-myc, HCC-22-5, p62, HCA25a, HER2, Hsp40, and Cyclin B1. Results: The positive rates of autoantibodies against RalA (24%), Hsp70 (22%), and P90 (20%) were the highest. Among 50 patients who showed seropositivity, 40 (80%) showed seropositivity for at least one antibody and 27 (54%) showed seropositivity for two or more antibodies. The positive rates of serum autoantibodies at each stage were as follows: 91% for stage I, 80% for stage II, and 67% for stage III. Conclusions: We developed ELISA systems to detect autoantibodies and successfully profiled 18 serum autoantibodies in Japanese patients with HCC. As pleural antibody responses were detected even in patients with stage I tumors, combination assay for these autoantibodies may be useful to detect early-stage HCC. Clinical trial registration number: UMIN 000014530.

Gastric cancer is the second leading cause of cancer death in the world, and effective diagnosis is extremely important for good outcome. We assessed the diagnostic potential of an autoantibody panel that may provide a novel tool for the early detection of gastric cancer. We analyzed data from patients with gastric cancer and normal controls in test and validation cohorts. Autoantibody levels were measured against a panel of six tumor-associated antigens (TAAs) by ELISA: p53, heat shock protein 70, HCC-22-5, peroxiredoxin VI, KM-HN-1, and p90 TAA. We assessed serum autoantibodies in 100 participants in the test cohort. The validation cohort comprised 248 participants. Autoantibodies to at least one of the six antigens showed a sensitivity/specificity of 49.0% (95% confidence interval [CI], 39.2-58.8%)/92.4% (95% CI, 87.2-97.6%), and 52.0% (95% CI, 42.2-61.8%)/90.5% (95% CI, 84.8-96.3%) in the test and validation cohorts, respectively. In the validation cohort, no significant differences were seen when patients were subdivided based on age, sex, depth of tumor invasion, lymph node metastasis, distant metastasis, peritoneal dissemination, or TNM stage. Patients who were positive for more than two antibodies in the panel tended to have a worse prognosis than those who were positive for one or no antibody. Measurement of autoantibody response to multiple TAAs in an optimized panel assay to discriminate patients with early stage gastric cancer from normal controls may aid in the early detection of gastric cancer.

Restoration of p53 functions is one of the therapeutic strategies for esophageal carcinoma which is often defective of the p53 pathway. We examined effects of CP-31398 which potentially increased expression of wild-type p53 or converted mutated p53 to the wild-type. We used 9 kinds of human squamous esophageal carcinoma cells with different p53 genotypes and examined expression of p53 and the related molecules in CP-31398-treated cells. Cisplatin, a DNA damaging agent, induced cleavages of PARP and caspase-3 without increase of p53 levels, indicating that the p53 down-stream pathway was disrupted in these cells. CP-31398 induced growth retardation but the cytotoxic effects were irrelevant to p53 genotype. CP-31398 influenced expression of p53 and the downstream molecules in a cell-dependent manner, but constantly increased p21 expression at the transcriptional level with decreased YY1 expression. Knockdown experiments with siRNA demonstrated that the CP-31398-mediated p21 up-regulation was unrelated with p53 expression but was associated with YY1 expression. We also showed that CP-31398-induced cell cycle changes including increase of G2/M populations was attributable to the up-regulated p21. These data collectively indicated that CP-31398 augmented endogenous p21 levels and induced cell cycle changes through regulation of YY1, and that YY1 was a novel target of CP-31398 in p53 dysfunctional cells.

Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

Background Mesothelioma patients often present with serosal effusions, which are ideal for cytopathological diagnoses. However, the morphological overlap between malignant and benign mesothelial proliferation can make a conclusive cytological diagnosis of mesothelioma elusive because immunohistochemical staining does not discriminate definitively between the two in this setting. p16 is deleted in up to 80% of pleural mesotheliomas. The aim of this study was to establish the correlation between the p16 deletion status of the cell block with that of its corresponding tumor using fluorescence in situ hybridization (FISH) analysis for individual patient tumors. Methods Twenty-two biopsies and 24 corresponding cell blocks, containing serosal effusions with atypical mesothelial cells from 22 patients with histologically confirmed pleural mesotheliomas, were analyzed with p16 FISH. Seventeen cell blocks consisting of serosal effusions with reactive mesothelial cells from nonmesothelioma cases were also analyzed. Combined immunofluorescence and FISH were also performed. Results Seventeen of the 22 mesothelioma patients (77.3%) showed homozygous deletions of p16 in the tumor tissue and in the atypical mesothelial cells from the cell blocks. p16 FISH followed by immunofluorescence with EMA was helpful towards identifying the mesothelioma cells in the cell blocks. Conclusion We confirmed that the p16 FISH results obtained from the cell blocks are as reliable as those from the tissue sections. Cell block analysis is recommended for patients with serosal effusions of unknown origins with the following methods: immunohistochemistry should be performed to validate the mesothelial origin, and p16 FISH should be performed to confirm malignancy. Diagn. Cytopathol. 2016;44:591-598. © 2016 Wiley Periodicals, Inc.

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes.

Cholesterol, a major component of the plasma membrane, determines the physical properties of biological membranes and plays a critical role in the assembly of membrane microdomains. Enrichment or deprivation of membrane cholesterol affects the activities of many signaling molecules at the plasma membrane. Cell detachment changes the structure of the plasma membrane and influences the localizations of lipids, including cholesterol. Recent studies showed that cell detachment changes the activities of a variety of signaling molecules. We previously reported that the localization and the function of the Src-family kinase Lyn are critically regulated by its membrane anchorage through lipid modifications. More recently, we found that the localization and the activity of Lyn were changed upon cell detachment, although the manners of which vary between cell types. In this review, we highlight the changes in the localization of Lyn and a role of cholesterol in the regulation of Lyn’s activation following cell detachment.

The Ras-like GTPases, RalA and RalB are members of the Ras superfamily of small GTPases. Aberrant activation of Ral is a major cause of human tumorigenesis induced by oncogenic Ras. Serum anti-RalA antibodies are induced in esophageal carcinoma patients. However, detailed comparisons of their pathological characteristics are unavailable, and conventional serum markers have not been well evaluated. Serum samples of 171 patients with esophageal squamous cell carcinoma and 73 healthy individuals were analyzed using specifically developed ELISA system for serum anti-RalA antibodies. A cut-off optical density value was fixed at 0.255 (the control mean + 2 SD). Clinicopathological characteristics and positive rates of conventional tumor markers were evaluated for seropositive patients. Overall positive rate for serum anti-RalA antibodies was 18 %, which gradually increased with the tumor stages. Although the positive rate for serum anti-RalA antibodies was comparable with that of carcinoembryonic antigen (24 %) and CYFRA21-1 (21 %), it was lower than the rate for serum p53 antibodies (31 %) and squamous cell carcinoma antigen (37 %). Although serum anti-RalA antibodies were not associated with other serum markers, it was inversely associated with serum p53 antibodies. No clear association was observed between serum anti-RalA antibodies and RalA immunoreactivity. Presence of serum anti-RalA antibodies is associated with tumor stages, but not with conventional tumor markers. Serum anti-RalA antibodies may be candidate serum markers in combination with other serum markers for esophageal squamous cell carcinoma.

Pancreatic carcinoma is relatively resistant to chemotherapy and cell death induced by replication of adenoviruses (Ad) can be one of the therapeutic options. Transduction efficacy of conventional type 5 Ad (Ad5) is however low and the cytotoxic mechanism by replication-competent Ad was not well understood. We constructed replication-competent Ad5 of which the E1A promoter region was replaced with a transcriptional regulatory region of the midkine, the survivin or the cyclooxygenase-2 gene, all of which were expressed at a high level in human tumors. We also prepared replication-competent Ad5 that were activated with the same region but had the type 35 Ad-derived fiber-knob region (AdF35) to convert the major cellular receptor for Ad infection from the coxsackie adenovirus receptor to CD46 molecules. Replication-competent AdF35 that were activated with the exogenous region produced cytotoxic effects on human pancreatic carcinoma cells greater than the corresponding Ad5 bearing with the same regulatory region. Cells infected with the AdF35 showed cytopathic effects and increased sub-G1 fractions. Caspase-9, less significantly caspase-8 and poly (ADP-ribose) polymerase, but not caspase-3 was cleaved and expression of molecules involved in autophagy and caspase-independent cell death pathways remained unchanged. Nevertheless, H2A histone family member X molecules were phosphorylated, and N-acetyl-L-cystein, an inhibitor for reactive oxygen species, suppressed the AdF35-mediated cytotoxicity. These data indicated a novel mechanism of Ad-mediated cell death and suggest a possible clinical application of the fiber-knob modified Ad.

We examined cytotoxicity of replication-competent type 5 adenoviruses (Ad5) in human pancreatic carcinoma cells with a p53-defective genotype. The replication-competent Ad5 of which E1A gene was activated by exogenous transcriptional regulatory sequences, derived from the midkine and survivin genes, achieved cytotoxicity to the pancreatic carcinoma. These cells were susceptible to replication-incompetent Ad5 expressing the wild-type p53 gene. We also produced the replication-competent Ad5 bearing the same exogenous regulatory sequences and the type 35 Ad-derived fiber-knob region, and showed that the cytotoxicity was comparable to that of the replication-competent Ad5 prototype. We then investigated possible combinatory effects of the fiber-modified replication-competent Ad and Ad5 expressing the wild-type p53 gene, both of which did not interfere respective infections. The combination produced synergistic cytotoxic effects with enhanced cleavages of caspase-3 and PARP molecules, and with increased sub-G1 fractions and annexin V-positive populations although the viral production of the replication-competent Ad was rather suppressed by expressed p53. Pancreatic cells infected with both Ad showed increase of p53 and decrease of MDM2 and p21 levels, compared with those infected with Ad expressing the p53 gene. These data collectively indicated that replication-competent Ad augmented susceptibility of pancreatic cells to apoptosis through upregulated p53 expression.

Type 5 adenoviruses expressing mda-7 gene (Ad-mda-7) induced cell death in various kinds of human tumors, but pancreatic carcinoma cells were relatively resistant to Ad-mda-7-mediated cytotoxicity. We then examined whether infection of Ad-mda-7 together with replication-competent Ad produced combinatory cytotoxic effects. We prepared replication-competent Ad, defective of the E1B55kDa gene or activated by a transcriptional regulatory region of the midkine or the survivin gene of which the expression was up-regulated in human tumors. Type 5 Ad bearing the exogenous regulatory region were further modified by replacing the fiber-knob region with that of type 35 Ad. Pancreatic carcinoma cells were infected with replication-incompetent Ad-mda-7 and the replication-competent Ad. Combinatory effects were examined with the CalcuSyn software and cell cycle analyses. Ad-mda-7 and the replication-competent Ad achieved cytotoxicity to pancreatic carcinoma. A combinatory use of Ad-mda-7 and either Ad defective of the E1B55kDa gene or Ad activated by the regulatory region produced synergistic cytotoxic effects. Cell cycle analyses demonstrated that the combination increased sub-G1 populations. These data collectively suggest that expression of MDA-7 augments cytotoxicity of replication-competent Ad and achieves adjuvant effects on Ad-mediated cell death.

Although serum p53 autoantibodies (s-p53-Abs) are induced even in the early stages of colorectal cancer, their positive rate is only approximately 20%. Therefore, we assessed the possibility of using other serum autoantibodies to increase the positive rates for detecting colorectal cancer. Autoantibodies against 17 tumor antigens (p53, RalA, HSP70, Galectin1, KM-HN-1, NY-ESO-1, p90, Sui1, HSP40, CyclinB1, HCC-22-5, c-myc, PrxVI, VEGF, HCA25a, p62, and Annexin II) were evaluated in 279 patients with colorectal cancer and 74 healthy controls. Cutoff values were fixed at mean + 3 standard deviations of serum titers in healthy controls. Autoantibodies with the highest positive rates were p53 (20%), RalA (14%), HSP70 (12%), and Galectin1 (11%). Combination assays using multiple autoantibodies increased the positive rates based on the number of autoantibodies used. Positive rates of 56, 62, 66, 71, and 73% were obtained with 6, 9, 11, 14, and 17 antibodies, respectively, for the overall disease. Moreover, these autoantibodies showed relatively high positive rates even during stage 0/I disease (55 and 70% with 6 and 17 antibodies, respectively). The measurement of set of 17 autoantibodies allowed autoantibody profiling in patients with colorectal cancer. The combination assay of six tumor antigens (p53, RalA, HSP70, Galectin1, KM-HN-1, and NY-ESO-1) achieved a positive rate of 56%. Such high positive rates will be helpful for detecting colorectal cancer regardless of tumor stages.

Background Optimizing targeting strategies for vectors in order to enhance antitumor activity and secure patient safety is important for cancer gene therapy. We previously identified two pancreatic cancer-targeting ligands (PFWSGAV: PFW and SYENFSA: SYE) by screening an adenovirus library in vivo and in vitro, respectively. Materials and methods To examine clinical usefulness, we assessed gene-transduction efficiency using surgically-resected pancreatic cancer specimens and ascites cells. Results For surgical specimens, vectors displaying PFW and SYE improved transduction efficiency by 4.4- and 4.3-fold, respectively. The SYE-displaying vector was >2-fold more efficient for all seven cases, whereas the PFW-displaying vector increased efficiency in two out of four cases. For ascites samples, both vectors increased gene-transduction efficiency of epithelial cell adhesion molecule (EpCAM)-positive ascites cells by >2-fold in two out of five cases. Conclusion Both vectors enhanced adenovirus infectivity of pancreatic cancer cells and have potential for gene therapy of pancreatic cancer; therefore they should be further evaluated in clinical studies.

Background Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma. Methods We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53. Results Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells. Conclusion These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3300-y) contains supplementary material, which is available to authorized users.

The addition of a targeting strategy is necessary to enhance oncolysis and secure safety of a conditionally replicative adenovirus (CRAd). We have constructed an adenovirus library displaying random peptides on the fiber, and have successfully identified a pancreatic cancer-targeting ligand (SYENFSA). Here, the usefulness of cancer-targeted CRAd for pancreatic cancer was examined as a preclinical study. First, we constructed a survivin promoter-regulated CRAd expressing enhanced green fluorescent protein gene (EGFP), which displayed the identified targeting ligand (AdSur-SYE). The AdSur-SYE resulted in higher gene transduction efficiency and oncolytic potency than the untargeted CRAd (AdSur) in several pancreatic cancer cell lines. An intratumoral injection of AdSur-SYE significantly suppressed the growth of subcutaneous tumors, in which AdSur-SYE effectively proliferated and spread. An ectopic infection in adjacent tissues and organs of intratumorally injected AdSur-SYE was decreased compared with AdSur. Then, to examine whether the targeting ligand actually enhanced the infectivity of CRAd in human pancreatic cancer tissues, tumor cells prepared from surgical specimens were infected with viruses. The AdSur-SYE increased gene transduction efficiency 6.4-fold higher than did AdSur in single cells derived from human pancreatic cancer, whereas the infectivity of both vectors was almost the same in the pancreas and other cancers. Immunostaining showed that most EGFP(+) cells were cytokeratin-positive in the sliced tissues, indicating that pancreatic cancer cells but not stromal cells were injected with AdSur-SYE. AdSur-SYE resulted in a stronger oncolysis in the primary pancreatic cancer cells co-cultured with mouse embryonic fibroblasts than AdSur did. CRAd in combination with a tumor-targeting ligand is promising as a next-generation of oncolytic virotherapy for pancreatic cancer.

Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma.

Dr Masatoshi Tagawa graduated with MD from the School of Medicine, Chiba University (Chiba, Japan) and completed the graduated course in Chiba University (awarded PhD) in 1984. He spent a few years in Stanford University (CA, USA) as a postdoctoral fellow and became an assistant professor at Chiba University. He then moved to Chiba Cancer Center Research Institute (Chiba, Japan) as the Head in Division of Pathology and Cell Therapy, and became a professor of Graduate School of Medicine at Chiba University. He is currently a council member of the Japan Society of Gene Therapy and International Society of Cell and Gene Therapy of Cancer. He also serves as an editor of Cancer Gene Therapy. His primary research field is molecular biology and oncology and he is currently working on mesothelioma at preclinical and clinical research levels.

Although it is now well known that some diseased areas, such as cancer nests, inflammation loci, and infarction areas, are acidified, little is known about cellular signal transduction, gene expression, and cellular functions under acidic conditions. Our group showed that different signal proteins were activated under acidic conditions compared with those observed in a typical medium of around pH 7.4 that has been used until now. Investigations of gene expression under acidic conditions may be crucial to our understanding of signal transduction in acidic diseased areas. In this study, we investigated gene expression in mesothelioma cells cultured at an acidic pH using a DNA microarray technique. After 24 h culture at pH 6.7, expressions of 379 genes were increased more than twofold compared with those in cells cultured at pH 7.5. Genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors numbered 35, 32, and 17 among the 379 genes, respectively. Since the functions of 78 genes are unknown, it can be argued that cells may have other genes for signaling under acidic conditions. The expressions of 37 of the 379 genes were observed to increase after as little as 2 h. After 24 h culture at pH 6.7, expressions of 412 genes were repressed more than twofold compared with those in cells cultured at pH 7.5, and the 412 genes contained 35, 76, and 7 genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors, respectively. These results suggest that the signal pathways in acidic diseased areas are different, at least in part, from those examined with cells cultured at a pH of around 7.4.

Malignant mesothelioma, developed in the thoracic cavity, is resistant to current treatments. Suppression of the local tumor growth is beneficial to the patients since mesothelioma infrequently metastasizes to extrapleural organs. A majority of the tumors have a homologous genetic deletion at the INK4A/ARF locus that includes the p14ARF and the p16INK4A genes, and the genetic defect results in an inactivation of the p53-mediated pathways and in progression of cell cycle through pRb phosphorylation. Preclinical studies targeting the genetic abnormality with adenoviruses showed that restoration of the p53 pathways induced pRb dephosphorylation and subsequently produced anti-tumor effects. A number of preclinical studies with different genes and vector systems demonstrated the therapeutic efficacy and raised the possibility of gene therapy in clinical settings. An intrapleural administration of vectors has several advantages in transducing pleural mesothelioma but activates rapid antibody production which impedes further gene expression. There have been several clinical studies conducted for mesothelioma and these trials showed the feasibility of intrapleural administrations of adenovirus vectors. In this review we summarize major preclinical and clinical gene therapy for mesothelioma, and discuss the advantages of gene therapy in the context of stimulating host immune systems. Accumulating clinical data suggest that an intrapleural administration of viral vectors has distinct aspects which are not observed in other administration routes.

The distinction between sarcomatoid mesothelioma and fibrous pleuritis is difficult based on histology, especially when the amount of tumor tissue examined via biopsy is small and immunohistochemical examination is inconclusive. We studied the usefulness of deletion of p16 with fluorescence in situ hybridization (FISH) and p16 hypermethylation with polymerase chain reaction for the diagnosis and prognosis of malignant pleural mesothelioma (MPM). We analyzed 50 MPMs, including 22 sarcomatoid mesothelioma cases and 10 fibrous pleuritis cases. We set the cutoff value of homozygous deletion pattern as 14.4% based on FISH signaling patterns using samples of fibrous pleuritis. The percentage of homozygous deletion pattern was higher than 14.4% in 55.6% of the epithelioid mesotheliomas (10/18) and in all of the sarcomatoid mesotheliomas (22/22). Methylation of p16 was observed in 7 (20.6%) of 34 informative cases. p16 FISH analysis can be a reliable test for distinguishing between sarcomatoid mesothelioma and fibrous pleuritis and a prognostic factor for MPM.

Introduction: Genetic characterization of malignant mesothelioma shows a homozygous deletion of the INK4A/ARF locus, which results in inactivation of the p53 pathways. Methods: We examined possible antitumor effects of adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55) on mesothelioma and investigated combinatory actions with the first-line chemotherapeutic agents. Results: Ad-delE1B55 produced cytotoxicity on mesothelioma cells, which was associated with p53 phosphorylation, pRb dephosphorylation, and cleavage of caspases. Ad-delE1B55–infected cells displayed hyperploidy at the cell-cycle analysis and showed enlarged nuclear configurations. Combination of Ad-delE1B55 plus cisplatin or pemetrexed produced antitumor effects in vitro. Furthermore, Ad-delE1B55 and cisplatin showed combinatory effects in an orthotopic animal model. Conclusions: Cell death caused by Ad-delE1B55 is attributable to cell-cycle arrest at M-phase checkpoint followed by activated apoptotic pathways, and combination of the first-line chemotherapeutic agents and the oncolytic adenovirus is a potential therapeutic for mesothelioma.

Malignant mesothelioma is a highly aggressive neoplasm, and the histologic subtype is one of the most reliable prognostic factors. Some biphasic mesotheliomas are difficult to distinguish from epithelioid mesotheliomas with atypical fibrous stroma. The aim of this study was to analyze p16/CDKN2A deletions in mesotheliomas by fluorescence in situ hybridization (FISH) and BAP1 immunohistochemistry to evaluate their potential role in the diagnosis of biphasic mesothelioma. We collected 38 cases of pleural mesotheliomas. The results of this study clearly distinguished 29 cases of biphasic mesothelioma from 9 cases of epithelioid mesothelioma. The proportion of biphasic mesotheliomas with homozygous deletions of p16/CDKN2A in total was 96.6% (28/29). Homozygous deletion of p16/CDKN2A was observed in 18 (94.7%) of 19 biphasic mesotheliomas with 100% concordance of the p16/CDKN2A deletion status between the epithelioid and sarcomatoid components in each case. Homozygous deletion of the p16/CDKN2A was observed in 7 (77.8%) of 9 epithelioid mesotheliomas but not in fibrous stroma. BAP1 loss was observed in 5 (38.5%) of 13 biphasic mesotheliomas and in both epithelioid and sarcomatoid components. BAP1 loss was observed in 5 (62.5%) of 8 epithelioid mesotheliomas but not in fibrous stroma. Homozygous deletion of p16/CDKN2A is common in biphasic mesotheliomas, and the analysis of only one component of mesothelioma is sufficient to show that the tumor is malignant. However, compared with histology alone, FISH analysis of the p16/CDKN2A status and BAP1 immunohistochemistry in the spindled mesothelium provide a more objective means to differentiate between biphasic mesothelioma and epithelioid mesothelioma with atypical stromal cells.

Esophageal squamous cell carcinoma (SCC) is frequently associated with a high mortality rate as a result of late diagnosis and/or aggressive behavior. Although multimodal treatment is applied for advanced tumors, many patients suffer from progressive disease and rapid recurrence. Besides various imaging techniques, serum biomarkers should also be useful for early diagnosing and treatment monitoring. A comprehensive review is provided here mainly on advancements of our clinical research in serum biomarkers to diagnose and/or monitor esophageal SCC. First, we focused on conventional secretory type serum markers, SCC-antigen and CYFRA 21-1. Both serum markers are useful to predict high-risk patients to develop recurrent disease. Second, we reviewed the clinicopathological significance of various angiogenic factors, vascular endothelial growth factor, thymidine phosphorylase, fibroblast growth factor, midkine, and hepatocyte growth factor. These growth factors could be useful biomarkers to predict lymph node and/or distant metastases. Finally, we reviewed advancements of clinical research on autoantibodies against tumor-specific antigens, particularly focused on serum p53 antibody. Because serum antibodies frequently respond to a small volume of tumors, they are useful in early tumor detection and prediction of residual cancer cells. Serum biomarkers may be a useful tool in the management of esophageal SCC.

Background Although replication-competent viruses have been developed for treating cancers, their cytotoxic effects are insufficient as a result of infection inhibited by the generation of neutralizing antibodies, and systemic administration is difficult as a result of the life-threatening serious side-effects of virus-induced cytokine surge. To overcome these critical problems, we devised a plasmid/polycation/polyanion complex and assessed the potential of ternary plasmid complexes coated with chondroitin sulfate in gene therapy for ovarian cancer. The antitumor effects of chondroitin sulfate-coated complex as an anionic component were compared with those of hyaluronic acid on ovarian cancer. Methods Plasmid harboring the gene of murine granulocyte macrophage-colony-stimulating factor (mGM-CSF) was complexed with polyethyleneimine (PEI) and hyaluronic acid or chondroitin sulfate. Murine ovarian cancer cells were injected into (C57BL/6 × C3H/He) F1 mice to prepare a subcutaneous or intraperitoneal tumor model. Results DNA/PEI was charged positively and DNA/PEI/chondroitin sulfate or DNA/PEI/hyaluronic acid was charged negatively. Plasmid-green fluorescent protein (GFP)/PEI coated with 10-kilodalton (kDa) chondroitin sulfate increased transfection efficiency compared to coating with chondroitin sulfate of higher-molecular-weight or hyaluronic acid. The transfection efficiency of GFP/PEI/10-kDa chondroitin sulfate in ovarian cancer cells was six-fold higher than that in normal cells. Intraperitoneal injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate prolonged survival compared to that coated with hyaluronic acid. Intratumoral injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate achieved mouse survival rates of 100%, although that with hyaluronic acid did not. Conclusions These findings suggest that GM-CSF/PEI coated with 10-kDa chondroitin sulfate has the potential for use in gene therapy of ovarian cancer. Copyright © 2012 John Wiley & Sons, Ltd.

Expression of human interleukin (IL)-24 in tumors achieved anti-tumor effects through apoptosis. IL-24 also induced secretion of proinflammatory cytokines, suggesting the role in immunity. We showed that murine IL-24 transcripts started from the second initiation codon and that expressed mIL-24 in tumors failed to induce apoptosis. Proliferation of murine cells expressing mIL-24 was the same as that of the parent cells and inoculation of the mIL-24-expressing tumors into syngeneic mice did not produce anti-tumor effects. Secretory mIL-24 did not induce the expression of the IL-6, TNF-α or IFN-γ gene in spleen cells. Expression of mIL-24 receptor subunits, IL-22R and IL-20R1, was undetectable in spleen cells even though they were stimulated by anti-CD3, anti-CD40 antibody or concanavalin A. Transduction of murine tumors with adenoviruses expressing the human IL-24 gene however suppressed the viability and decreased the tumor growth. These data suggest that mIL-24 is functionally irrelevant to the human counterpart.

The novel human gene family encoding neuronal leucine rich repeat (NLRR) proteins were identified as prognostic markers from our previous screening of primary neuroblastoma (NB) cDNA libraries. Of the NLRR gene family members, NLRR1 and NLRR3 are associated with the regulation of cellular proliferation and differentiation, respectively. However, the functional regulation and clinical significance of NLRR2 in NB remain unclear. Here, we evaluated the differential expression of NLRR2, where high expressions of NLRR2 were significantly associated with a poor prognosis of NB (P = 0.0009), in 78 NBs. Enforced expression of NLRR2 in NB cells enhanced cellular proliferation and induced resistance to retinoic acid (RA)-mediated cell growth inhibition. In contrast, knockdown of NLRR2 exhibited growth inhibition effects and enhanced RA-induced cell differentiation in NB cells. After RA treatment, NLRR2 expression was increased and correlated with the upregulation of c-Jun, a member of the activator protein-1 (AP-1) family in NB cells. Moreover, the expressions of NLRR2 and c-Jun were suppressed by treatment with a JNK inhibitor, which ameliorated the promoter activity of the NLRR2 gene while knockdown of c-Jun reduced NLRR2 expression. We then searched AP-1 binding consensus in the NLRR2 promoter region and confirmed c-Jun recruitment at a consensus. Conclusively, NLRR2 must be an inducible gene regulated by the JNK pathway to enhance cell survival and inhibit NB cell differentiation. Therefore, NLRR2 should have an important role in NB aggressiveness and be a potential therapeutic target for the treatment of RA resistant and aggressive NB.