Combined treatment with epigenetic agents enhances anti-tumor activity of MAGE-D4 peptide-specific T cells by upregulating the MAGE-D4 expression in glioma.

Objective: The study evaluated the efficacy of combined epigenetic drugs of decitabine (DAC), valproic acid (VPA), and trichostatin A (TSA) on immunotherapy against glioma. Methods: The expression and prognosis of MAGE-D4 in glioma were analyzed online, and the expression of MAGE-D4 and HLA-A2 in glioma induced by epigenetic drugs was detected by qRT-PCR, Western blot, and flow cytometry. The methylation status of the MAGE-D4 promoter was determined by pyrosequencing. An HLA-A2 restricted MAGE-D4 peptide was predicted and synthesized. An affinity assay and a peptide/HLA complex stability assay were performed to determine the affinity between peptide and HLA. CCK8 assay, CFSE assay, ELISA and ELISPOT were performed to detect the function of MAGE-D4 peptide-specific T cells. Flow cytometry, ELISA, and cytotoxicity assays were used to detect the cytotoxicity effect of MAGE-D4 peptide-specific T cells combined with epigenetic drugs against glioma in vitro. Finally, the glioma-loaded mouse model was applied to test the inhibitory effect of specific T cells on gliomas in vivo. Results: MAGE-D4 was highly expressed in glioma and correlated with poor prognosis. Glioma cells could be induced to express MAGE-D4 and HLA-A2 by epigenetic drugs. MAGE-D4-associated peptides were found that induce DCs to stimulate the highest T-cell activities of proliferation, IL-2 excretion, and IFN-γ secretion. MAGE-D4 peptide-specific T cells treated with TSA only or combining TSA and DAC had the most cytotoxicity effect, and its cytotoxicity effect on glioma cells decreased significantly after HLA blocking. In vivo experiments also confirmed that MAGE-D4-specific T cells inhibit TSA-treated glioma. Conclusion: MAGE-D4 is highly expressed in glioma and correlated with the prognosis of glioma. The novel MAGE-D4 peptide identified was capable of inducing MAGE-D4-specific T cells that can effectively inhibit glioma growth, and the epigenetic drug application can enhance this inhibition.

FMR1NB Involved in Glioma Tumorigenesis Is a Promising Target for Prognosis and Therapy.

OBJECTIVE: Cancer/testis antigen FMR1NB is aberrantly expressed in various types of cancer, but not in normal tissues except for testis. This study aimed to investigate the expression and functional role of FMR1NB in glioma. METHODS: The expression of FMR1NB mRNA and protein was determined using RT-PCR and immunohistochemistry, respectively, in glioma specimens from 83 patients at follow-up. The effects of siRNA-mediated FMR1NB silencing on malignant biological behaviors were evaluated in glioma cell lines A172 and U251. RESULTS: FMR1NB mRNA and protein expression was detected in 58.8% (77/131) and 46.34% (57/123) of glioma tissues, respectively. FMR1NB protein was positively correlated with World Health Organization grade and found to be an independent prognostic marker for poor outcome. Knockdown of FMR1NB induced apoptosis and suppressed proliferation, adhesion, migration, and invasion by modulating the expression of cyclin A, CDK2, caspase-3, E-cadherin, and N-cadherin in A172 and U251 cells. CONCLUSION: Our findings suggest that FMR1NB contributes to the tumorigenesis of glioma cells and may represent a potential prognostic biomarker and an attractive therapeutic target in glioma.

Combined treatment with epigenetic agents enhances anti-tumor activity of T cells by upregulating the ACRBP expression in hepatocellular carcinoma.

OBJECTIVE: To evaluate the efficacy of combined epigenetic drugs of decitabine (DAC), valproic acid (VPA) and trichostatin A (TSA) on immunotherapy with a murine model of hepatocellular carcinoma (HCC). METHODS: Dendritic cells (DCs) transduced with recombinant lentivirus expressing a cancer-testis antigen, acrosin binding protein (ACRBP), are referred to as DC/ACRBP. CD8+ T cells were harvested from spleens of C57BL/6 mice and activated by DC/ACRBP. Cytotoxicity of DC/ACRBP-activated T cells was analyzed by cytotoxicity and murine xenograft assays. RESULTS: Cytotoxicity assay results revealed that DC/ACRBP-activated T cells exhibited the highest cytotoxicity against HCC cells pre-treated with triple drugs (DAC+VPA+TSA) compared with dual drugs (DAC+VPA and DAC+TSA) and single drug (DAC, VPA and TSA) respectively. Analyses of RT-PCR and immunoblotting demonstrated that the highest ACRBP expression of HCC cells was induced by the triple drugs compared with the single and dual drugs. These results indicated that DC/ACRBP-activated T cells might be ACRBP-specific lymphocytes, and the augmented cytotoxicity may be dependent on the upregulation of ACRBP expression. These assumptions were further confirmed by xenograft tumor assay. Tumor cells of mice administrated with the triple drugs exhibited increased ACRBP expression compared with those of mice without administration. As expected, DC/ACRBP-activated T cells adopted by mice injected with the triple drugs, compared with those adopted by mice without injection, remarkably impeded growth and facilitated apoptosis of tumor cells. CONCLUSION: These data suggested that combined treatment with DAC, VPA and TSA may enhance the anti-tumor efficacy of ACRBP-specific T cells by upregulating ACRBP expression in HCC.

Identification of Dysregulated microRNAs in Glioma Using RNA-sequencing.

Glioma is the most common malignant brain tumor in central nervous system. Despite advances in the treatment of glioma such as surgery and chemoradiotherapy, most patients are easy to relapse, resulting in adverse clinical outcomes. Hence, effective molecular-targeting treatment may be one of attractive strategies for glioma therapy. The dysregulated microRNAs (miRNAs), one of the candidates of therapeutic targets, are believed to play an important role in the progression of glioma. In this study, we aimed to examine the expression profile of miRNAs in glioma and provide a reference for glioma therapy. Firstly, expression profile of miRNAs in 5 normal brain tissues, 5 low-grade glioma (LGG) tissues and 5 glioblastoma (GBM) tissues was detected by RNA sequencing (RNA-seq). Next, the target genes of differentially expressed miRNAs (DEmiRNAs) were predicted and then GO enrichment and KEGG pathway analysis performed by bioinformatics. Finally, 10 miRNAs which were significantly up- or down-regulated both in GBM and LGG were validated by real-time quantitative PCR (qRT-PCR). RNA-seq results indicated a number of DEmiRNAs in glioma. There were 64 up-regulated miRNAs and 17 down-regulated miRNAs in LGG, and 181 up-regulated miRNAs and 124 down-regulated miRNAs in GBM, respectively. Bioinformatics analysis showed that the target genes of these DEmiRNAs were enriched in various biological processes and signaling pathways such as cell metabolic and developmental process. Selected DEmiRNAs were further confirmed by qRT-PCR. miRNA-10b-5p, miRNA-92b-3p and miRNA-455-5p were significantly up-regulated in both GBM and LGG; while miRNA-542-3p was significantly up-regulated in LGG; miRNA-184 and miRNA-206 were significantly down-regulated in both GBM and LGG; miRNA-766-5p and miRNA-1-3p were significantly down-regulated in GBM. The subject of our study demonstrated several dysregulated miRNAs may serve as a potential therapeutic target for glioma.

Cancer-testis Antigen OY-TES-1 Expression and Immunogenicity in Hepatocellular Carcinoma.

Cancer testis (CT) antigens have received particular attention in cancer immunotherapy. OY-TES-1 is a member of CT antigens. This study was to evaluate OY-TES-1 expression and immunogenicity in hepatocelluar carcinoma (HCC). OY-TES-1 mRNA expression was detected in 56 HCC tissues and 5 normal liver tissues by reverse transcriptase PCR (RT-PCR). Of the 56 cases of HCC tissues tested, 37 cases had tumor and matched adjacent non-cancer tissues and were subjected to both RT-PCR and quantitative real-time PCR. OY-TES-1 protein was subsequently observed on a panel of tissue microarrays. Sera from patients were tested for OY-TES-1 antibody by ELISA. To identify OY-TES-1 capable of inducing cellular immune response, OY-TES-1 protein was used to sensitize dentritic cells and the cytotoxicity effect was measured in vitro. The results showed that OY-TES-1 mRNA was highly expressed in 41 of the 56 (73.21%) HCC tissues, whereas none in 5 normal liver tissues. OY-TES-1 mRNA was frequently expressed not only in HCC tissues (72.97%, 27/37), but also in paired adjacent non-cancer tissues (64.86%, 24/37). But the mean expression level of OY-TES-1 mRNA in HCC tissues was significantly higher than that in adjacent non-cancer tissues (0.76854 vs. 0.09834, P=0.021). Immunohistochemistry showed that OY-TES-1 protein expression was detected in 6 of the 49 cases of HCC tissues, and absent in 9 cases of normal liver and 6 cases of cirrhosis tissues. Seropositivity was detected in 10 of the 45 HCC patients, but not detected in 17 cirrhosis patients and 76 healthy donors. The specific cytotoxic T cells elicited by OY-TES-1 could kill HLA-A2+ HCC cell line which expressed OY-TES-1. The target lysis was mainly HLA class I -dependent and could be blocked by antibodies against monomorphic HLA class I but not HLA class II molecule. In summary, OY-TES-1 expression is up-regulated in HCC tissues and can be recognized by humoral and cellular responses, which suggests that OY-TES-1 is an attractive target for tumor immunotherapy in HCC.

MAGED4 expression in glioma and upregulation in glioma cell lines with 5-aza-2'-deoxycytidine treatment.

Melanoma-associated antigen (MAGE) family genes have been considered as potentially promising targets for anticancer immunotherapy. MAGED4 was originally identified as a glioma-specific antigen. Current knowledge about MAGED4 expression in glioma is only based on mRNA analysis and MAGED4 protein expression has not been elucidated. In the present study, we investigated this point and found that MAGED4 mRNA and protein were absent or very lowly expressed in various normal tissues and glioma cell line SHG44, but overexpressed in glioma cell lines A172,U251,U87-MG as well as glioma tissues, with significant heterogeneity. Furthermore, MAGED4 protein expression was positively correlated with the glioma type and grade. We also found that the expression of MAGED4 inversely correlated with the overall methylation status of the MAGED4 promoter CpG island. Furthermore, when SHG44 and A172 with higher methylation were treated with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) reactivation of MAGED4 mRNA was mediated by significant demethylation in SHG44 instead of A172. However, 5-AZA-CdR treatment had no effect on MAGED4 protein in both SHG44 and A172 cells. In conclusion, MAGED4 is frequently and highly expressed in glioma and is partly regulated by DNA methylation. The results suggest that MAGED4 might be a promising target for glioma immunotherapy combined with 5-AZA-CdR to enhance its expression and eliminate intratumor heterogeneity.