Evaluation of heterogeneous metabolic profile in an orthotopic human glioblastoma xenograft model using compressed sensing hyperpolarized 3D 13C magnetic resonance spectroscopic imaging.

High resolution compressed sensing hyperpolarized (13)C magnetic resonance spectroscopic imaging was applied in orthotopic human glioblastoma xenografts for quantitative assessment of spatial variations in (13)C metabolic profiles and comparison with histopathology. A new compressed sensing sampling design with a factor of 3.72 acceleration was implemented to enable a factor of 4 increase in spatial resolution. Compressed sensing 3D (13)C magnetic resonance spectroscopic imaging data were acquired from a phantom and 10 tumor-bearing rats following injection of hyperpolarized [1-(13)C]-pyruvate using a 3T scanner. The (13)C metabolic profiles were compared with hematoxylin and eosin staining and carbonic anhydrase 9 staining. The high-resolution compressed sensing (13)C magnetic resonance spectroscopic imaging data enabled the differentiation of distinct (13)C metabolite patterns within abnormal tissues with high specificity in similar scan times compared to the fully sampled method. The results from pathology confirmed the different characteristics of (13)C metabolic profiles between viable, non-necrotic, nonhypoxic tumor, and necrotic, hypoxic tissue.

Epigenetic aberrations and therapeutic implications in gliomas.

Almost all cancer cells have multiple epigenetic abnormalities, which combine with genetic changes to affect many cellular processes, including cell proliferation and invasion, by silencing tumor-suppressor genes. In this review, we focus on the epigenetic mechanisms of DNA hypomethylation and CpG island hypermethylation in gliomas. Aberrant hypermethylation in promoter CpG islands has been recognized as a key mechanism involved in the silencing of cancer-associated genes and occurs at genes with diverse functions related to tumorigenesis and tumor progression. Such promoter hypermethylation can modulate the sensitivity of glioblastomas to drugs and radiotherapy. As an example, the methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter is a specific predictive biomarker of tumor responsiveness to chemotherapy with alkylating agents. Further, we reviewed reports on pyrosequencing - a simple technique for the accurate and quantitative analysis of DNA methylation. We believe that the quantification of MGMT methylation by pyrosequencing might enable the selection of patients who are most likely to benefit from chemotherapy. Finally, we also evaluated the potential of de novo NY-ESO-1, the most immunogenic cancer/testis antigen (CTA) discovered thus far, as an immunotherapy target. The use of potent epigenetics-based therapy for cancer cells might restore the abnormally regulated epigenomes to a more normal state through epigenetic reprogramming. Thus, epigenetic therapy may be a promising and potent treatment for human neoplasia.

Retrovirally engineered T-cell-based immunotherapy targeting type III variant epidermal growth factor receptor, a glioma-associated antigen.

The isotype of epidermal growth factor receptor variant III (EGFRvIII) is often identified in glioblastomas. Previously, we created a mouse monoclonal antibody, 3C10 (IgG2b), that specifically recognized EGFRvIII, and a recombinant single-chain variable fragment of 3C10. The aim of the current study was to develop genetically engineered T cells, termed T-bodies, that express a chimeric receptor consisting of the 3C10 single-chain variable fragment coupled to signaling modules such as the CD3zeta (ζ) chain, for the treatment of tumors expressing mutant EGFR. After successful construction of the chimeric 3C10/CD3ζ T-cell receptor, its expression on the T-body was observed using western blotting and flow cytometry. The specificity of the T-body for EGFRvIII was evaluated using an interferon-gamma Elispot assay and a standard (51) Cr-release cytotoxicity assay. Furthermore, we demonstrated that the systemically delivered T-body infiltrated the intrabrain tumor and significantly delayed tumor growth. These results indicate that the T-body expressing the chimeric 3C10/CD3ζ T-cell receptor specifically recognized glioma cells expressing EGFRvIII. In conclusion, T-body-based immunotherapy appears to be a promising approach for the treatment of glioma.

Interferon-beta, MCNU, and conventional radiotherapy for pediatric patients with brainstem glioma.

BACKGROUND: Most children with brainstem glioma die within 2 years of diagnosis, and the median survival time for patients with this condition is less than 1 year. The role of chemotherapy in the treatment of children with brainstem glioma is not well defined. The primary aim of this study is to evaluate the effects of treatment with interferon-beta (IFN-beta), ranimustine (MCNU), and radiotherapy (IMR therapy) administered to brainstem glioma patients treated at our institution. We also determined patient response to IMR therapy by evaluating O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in serum DNA. PROCEDURES: We retrospectively reviewed 15 patients who were newly diagnosed to have brainstem tumors and were administered IFN-beta (1-2 MIU/day, days 1-7; 0.5-1 MIU/day, days 8-14) and MCNU (80 mg/m(2) on day 2) concurrently with conventional radiotherapy. Responses were assessed by MRI scan, and data on clinical course and toxicity were obtained from the medical records. The MGMT promoter methylation in serum DNA of five patients was assayed by methylation-specific PCR. RESULTS: Of the 15 patients, partial response, stable disease, and progressive disease were noted in 5 patients each. The median overall survival time and the median progression-free survival time were 14.7 and 4.6 months, respectively. The protocol was not terminated in any of the patients because of hematological toxicity, nephrotoxicity, or neurotoxicity. The MGMT promoter methylation status in the serum appeared to correlate with a positive response to IMR therapy. CONCLUSIONS: The IMR combination therapy is well tolerated and may be a promising treatment for brainstem glioma.

Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) most commonly arises from chronic inflammation due to viral infection, as a result of genetic and epigenetic abnormalities. A global picture of epigenetic changes in HCC is lacking. We used methylated CpG island amplification microarrays (MCAMs) to study 6458 CpG islands in HCC and adjacent preneoplastic tissues [chronic hepatitis (CH) or liver cirrhosis (LC)] in comparison with normal liver tissues where neither viral infection nor hepatitis has existed. MCAM identified 719 (11%) prominent genes of hypermethylation in HCCs. HCCs arising from LC had significantly more methylation than those arising from CH (1249 genes or 19% versus 444 genes or 7%, P < 0.05). There were four patterns of aberrant methylation: Type I (4%, e.g. matrix metalloproteinase 14) shows a substantially high methylation level in adjacent tissue and does not increase further in cancer. Type II (55%, e.g. RASSF1A) shows progressively increasing methylation from adjacent tissue to HCC. Type III (4%, e.g. GNA14) shows decreased methylation in adjacent tissue but either similar or increased methylation in HCC. Type IV (37%, e.g. CDKN2A) shows low levels of methylation in normal tissue and adjacent tissue but high levels in HCC. These DNA methylation changes were confirmed by quantitative pyrosequencing methylation analysis in representative 24 genes and were analyzed for correlation with clinicopathological parameters in 38 patients. Intriguingly, methylation in the Type IV genes is characteristic of moderately/poorly differentiated cancer. Our global epigenome analysis reveals distinct patterns of methylation that are probably to represent different pathophysiologic processes in HCCs.

The DNA demethylating agent 5-aza-2'-deoxycytidine activates NY-ESO-1 antigenicity in orthotopic human glioma.

Cancer/testis antigens (CTAs) are considered to be suitable targets for the immunotherapy of human malignancies. It has been demonstrated that in a variety of tumors, the expression of certain CTAs is activated via the demethylation of their promoter CpG islands. In our study, we have shown that while the composite expression of 13 CTAs in 30 human glioma specimens and newly established cell lines from the Japanese population was nearly imperceptible, the DNA-demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) markedly reactivated CTA expression in glioma cells but not in normal human cells. We quantified the diminished methylation status of NY-ESO-1-one of the most immunogenic CTAs-following 5-aza-CdR treatment by using a novel Pyrosequencing technology and methylation-specific PCR. Microarray analysis revealed that 5-aza-CdR is capable of signaling the immune system, particularly, human leukocyte antigen (HLA) class I upregulation. (51)Cr-release cytotoxicity assays and cold target inhibition assays using NY-ESO-1-specific cytotoxic T lymphocyte (CTL) lines demonstrated the presentation of de novo NY-ESO-1 antigenic peptides on the cell surfaces. In an orthotopic xenograft model, the systemic administration of 5-aza-CdR resulted in a significant volume reduction of the transplanted tumors and prolonged the survival of the animals after the adoptive transfer of NY-ESO-1-specific CTLs. These results suggested that 5-aza-CdR induces the expression of epigenetically silenced CTAs in poorly immunogenic gliomas and thereby presents a new strategy for tumor immunotherapy targeting 5-aza-CdR-induced CTAs.

A combination of IFN-beta and temozolomide in human glioma xenograft models: implication of p53-mediated MGMT downregulation.

PURPOSE: Methylation of the O(6)-methyguanine-DNA methyltransferase (MGMT) gene promoter in gliomas has been reported to be a useful predictor of the responsiveness to temozolomide (TMZ). In our previous experiments, we observed that IFN-beta sensitized TMZ-resistant glioma cells with the unmethylated MGMT promoter and that the mechanism of action was possibly due to attenuation of MGMT expression via induction of TP53. In this study, (1) we explored the synergistic effect of IFN-beta and TMZ in the animal model, and (2) clarified the role of IFN-beta induced TP53 in the human MGMT promoter. METHODS: (1) Nude mice with either subcutaneous T98 (TMZ-resistant) or U251SP (TMZ-sensitive) tumor were treated with IFN-beta/TMZ for 5 consecutive days. (2) The MGMT promoter activity was assayed by a luciferase reporter system in Saos2 (p53-null) cells transduced with a p53-adenoviral vector, and T98 glioma cells treated with IFN-beta. RESULTS: (1) A combination of IFN-beta/TMZ had significant synergistic antitumor activity on the growth of both T98 and U251SP tumors. (2) MGMT promoter activity was suppressed by either adenovirally transduced p53 or IFN-beta. CONCLUSIONS: It would be appealing to consider a prospective clinical trial in which genetic markers are used for personalized drug selection, eliciting other forms of treatment or inhibition of MGMT for those with MGMT expression. In this context, IFN-beta inactivates MGMT via p53 gene induction and enhances the therapeutic efficacy to TMZ.

Identification of a human leukocyte antigen-A24-restricted T-cell epitope derived from interleukin-13 receptor alpha2 chain, a glioma-associated antigen.

OBJECT: The human leukocyte antigen-A24 (HLA-A24) allele is highly expressed in Asians. This allele is expressed in 60% of the Japanese population and in a significant number of people of other ethnicities. The interleukin-13 type alpha2 receptor (IL-13Ralpha2) has been shown to be a glioma-specific antigen, and is abundantly expressed in a majority of high-grade astrocytomas. In this study, the authors first investigated the suitability of IL-13Ralpha2 as a target antigen of malignant glioma cells, and then identified a potential HLA-A24-restricted peptide derived from IL-13Ralpha2. METHODS: The expression of IL-13Ralpha2 in glioma tissues was examined by reverse transcription-polymerase chain reaction analysis. To identify the desired epitope, the authors selected 5 candidate peptides from IL-13Ralpha2 that were predicted to bind to HLA-A24. The lytic activity of cytotoxic T lymphocytes (CTLs) induced by peptide-pulsed dendritic cells was analyzed against various glioma cell lines and freshly isolated human glioma cells. RESULTS: In a series of glioma tissues obtained in 29 patients, the authors found that > 50% of high-grade gliomas expressed IL-13Ralpha2. Of the 5 peptides tested, P174 (WYEGLDHAL) was found to be the most useful for the induction of HLA-A24-restricted and IL-13Ralpha2-specific CTLs. A CTL line induced by P174 also showed antigen-specific cytotoxicity to surgically removed glioma cells depending on their level of expression of IL-13Ralpha2 and HLA-A24. CONCLUSIONS: Interleukin-13Ralpha2 is a glioma-specific antigen, and the immunogenic peptide P174 may contribute to a peptide-based immunotherapy against malignant glioma cells expressing HLA-A24.

Intravenously transplanted human neural stem cells migrate to the injured spinal cord in adult mice in an SDF-1- and HGF-dependent manner.

Neural stem cell (NSC) transplantation has exhibited considerable therapeutic potential in spinal cord injury. However, most experiments in animals have been performed by injecting these cells directly into the injured spinal cord. A cardinal feature of NSCs is their exceptional migratory ability through the nervous system. Based on the migratory ability of NSCs, we investigated whether minimally invasive intravenous delivery of NSCs could facilitate their migration to the injured spinal cord and identified the chemo-attractants secreted by the lesions. Nude mice were injected intravenously with labelled human NSCs at 3, 7 and 10 days after the compression of the spinal cord at the T8 level. The migration of NSCs to the lesioned spinal cord was highest at 7 days after injury; this correlated with the peak of hepatocyte growth factor and stromal cell-derived factor-1 mRNA expressions in the lesion but not with the disruption of the blood-brain barrier. Finally, the grafted NSCs differentiated into neuronal and glial subpopulations at 21 days after transplantation. Our study suggests that intravenously administered NSCs can be employed as a renewable source for replacing lost cells for the treatment of spinal cord injuries.

Malignant transformation-related genes in meningiomas: allelic loss on 1p36 and methylation status of p73 and RASSF1A.

OBJECT: Analysis of meningiomas supports the suggestion that loss of heterozygosity (LOH) of chromosome arm 1p plays an important role in malignancy. The aim of this study was to identify genes related to meningioma progression from the benign state to the atypical and anaplastic states by examining 1p LOH and the promoter methylation of RASSF1A and p73. METHODS: The authors studied 40 surgical specimens (22 WHO Grade I, 11 Grade II, and seven Grade III) obtained in 37 patients with meningioma. The LOH at 1p36 was analyzed using microsatellite markers, and promoter methylation of p73 and RASSFIA was analyzed using methylation-specific polymerase chain reaction. RESULTS: No 1p LOH was detected in the Grade I tumors, whereas it was detected in more than 80% of the Grade II and III tumors. Methylation of the p73 promoter was observed in 81.8 and 71.4% of the Grade II and III tumors, respectively, but it was not observed in any of the Grade I tumors; methylation of the RASSF1A promoter was observed in 18.2, 63.6, and 42.9% of the Grade I, II, and III tumors, respectively. Interestingly, 1p LOH and p73 promoter hypermethylation were detected in the malignantly transformed tumors but not in the lower-grade primary ones. CONCLUSIONS: Based on the hypothesis that meningiomas cumulatively acquire genetic alterations and thus progress from the benign to the atypical and anaplastic states, genetic alterations in the methylation status of p73 or RASSF1A along with 1p LOH may result in the malignant transformation of a meningioma. This type of genetic fingerprint may play both diagnostic and therapeutic roles.

Efficacy of temozolomide is correlated with 1p loss and methylation of the deoxyribonucleic acid repair gene MGMT in malignant gliomas.

Promoter methylation of the deoxyribonucleic acid (DNA) repair gene, O(6)-methylguanine-DNA methyltransferase (MGMT), is associated with improved outcome of patients with glioblastoma multiforme and anaplastic astrocytoma treated with temozolomide (TMZ). Molecular genetic analysis of loss of heterozygosity (LOH) of 1p, 19q, or 10q, p53 mutation, and MGMT promoter methylation was performed in 44 assessable tumor specimens obtained from 46 patients with recurrent malignant gliomas, including 21 with glioblastoma multiforme, 17 with anaplastic astrocytoma, and eight with anaplastic oligoastrocytoma, which have heterogeneous features and variable histological diagnosis, to assess the correlation with the response to TMZ. LOHs of 1p and 19q, and MGMT promoter methylation showed positive correlations with the clinical response to TMZ therapy (p < 0.005, 0.05, and 0.05, respectively; Fisher's exact test). In addition, LOH of 1p and MGMT promoter methylation were associated with longer progression-free survival (p < 0.05 and 0.05, respectively; Cox regression analysis). LOH of 1p in the heterogeneous population of malignant gliomas may be one of the important factors besides MGMT methylation that predict better outcome in patients treated with TMZ.

Changes in pyruvate metabolism detected by magnetic resonance imaging are linked to DNA damage and serve as a sensor of temozolomide response in glioblastoma cells.

Recent findings show that exposure to temozolomide (TMZ), a DNA-damaging drug used to treat glioblastoma (GBM), can suppress the conversion of pyruvate to lactate. To understand the mechanistic basis for this effect and its potential utility as a TMZ response biomarker, we compared the response of isogenic GBM cell populations differing only in expression of the DNA repair protein methyltransferase (MGMT), a TMZ-sensitivity determinant, after exposure to TMZ in vitro and in vivo. Hyperpolarized [1-((13))C]-pyruvate-based MRI was used to monitor temporal effects on pyruvate metabolism in parallel with DNA-damage responses and tumor cell growth. TMZ exposure decreased conversion of pyruvate to lactate only in MGMT-deficient cells. This effect coincided temporally with TMZ-induced increases in levels of the DNA-damage response protein pChk1. Changes in pyruvate to lactate conversion triggered by TMZ preceded tumor growth suppression and were not associated with changes in levels of NADH or lactate dehydrogenase activity in tumors. Instead, they were associated with a TMZ-induced decrease in the expression and activity of pyruvate kinase PKM2, a glycolytic enzyme that indirectly controls pyruvate metabolism. PKM2 silencing decreased PK activity, intracellular lactate levels, and conversion of pyruvate to lactate in the same manner as TMZ, and Chk1 silencing blocked the TMZ-induced decrease in PKM2 expression. Overall, our findings showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by MRI methods as an early sensor of TMZ therapeutic response.

Early Chk1 phosphorylation is driven by temozolomide-induced, DNA double strand break- and mismatch repair-independent DNA damage.

Temozolomide (TMZ) is a DNA methylating agent used to treat brain cancer. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair during DNA replication and trigger cycles of futile mismatch repair (MMR). Futile MMR in turn leads to the formation of DNA single and double strand breaks, Chk1 and Chk2 phosphorylation/activation, cell cycle arrest, and ultimately cell death. Although both pChk1 and pChk2 are considered to be biomarkers of TMZ-induced DNA damage, cell-cycle arrest, and TMZ induced cytotoxicity, we found that levels of pChk1 (ser345), its downstream target pCdc25C (ser216), and the activity of its upstream activator ATR, were elevated within 3 hours of TMZ exposure, long before the onset of TMZ-induced DNA double strand breaks, Chk2 phosphorylation/activation, and cell cycle arrest. Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status. Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells. These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.

Interferon-ß delivery via human neural stem cell abates glial scar formation in spinal cord injury.

Glial scar formation is the major impedance to axonal regrowth after spinal cord injury (SCI), and scar-modulating treatments have become a leading therapeutic goal for SCI treatment. In this study, human neural stem cells (NSCs) encoding interferon-ß (INF-ß) gene were administered intravenously to mice 1 week after SCI. Animals receiving NSCs encoding IFN-ß exhibited significant neurobehavioral improvement, electrophysiological recovery, suppressed glial scar formation, and preservation of nerve fibers in lesioned spinal cord. Systemic evaluation of SCI gliosis lesion site with lesion-specific microdissection, genome-wide microarray, and MetaCore pathway analysis identified upregulation of toll-like receptor 4 (TLR4) in SCI gliosis lesion site, and this led us to focus on TLR4 signaling in reactive astrocytes. Examination of primary astrocytes from TLR4 knockout mice, and in vivo inhibition of TLR4, revealed that the effect of IFN-ß on the suppression of glial scar formation in SCI requires TLR4 stimulation. These results suggest that IFN-ß delivery via intravenous injection of NSCs following SCI inhibits glial scar formation in spinal cord through stimulation of TLR4 signaling.

Chromatin regulator PRC2 is a key regulator of epigenetic plasticity in glioblastoma.

Tumor cell plasticity contributes to functional and morphologic heterogeneity. To uncover the underlying mechanisms of this plasticity, we examined glioma stem-like cells (GSC) where we found that the biologic interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of polycomb repressive complex 2 (PRC2), a complex that modifies chromatin structure. PRC2 mediates lysine 27 trimethylation on histone H3 and in GSC it affected pluripotency or development-associated genes (e.g., Nanog, Wnt1, and BMP5) together with alterations in the subcellular localization of EZH2, a catalytic component of PRC2. Intriguingly, exogenous expression of EZH2-dNLS, which lacks nuclear localization sequence, impaired the repression of Nanog expression under differentiation conditions. RNA interference (RNAi)-mediated attenuation or pharmacologic inhibition of EZH2 had little to no effect on apoptosis or bromodeoxyuridine incorporation in GSCs, but it disrupted morphologic interconversion and impaired GSC integration into the brain tissue, thereby improving survival of GSC-bearing mice. Pathologic analysis of human glioma specimens revealed that the number of tumor cells with nuclear EZH2 is larger around tumor vessels and the invasive front, suggesting that nuclear EZH2 may help reprogram tumor cells in close proximity to this microenvironment. Our results indicate that epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Thus, PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, offering a new paradigm for glioma treatment.

Long-term survival in patients with newly diagnosed primary central nervous system lymphoma treated with dexamethasone, etoposide, ifosfamide and carboplatin chemotherapy and whole-brain radiation therapy.

In the present study, we aimed to evaluate the safety and efficacy of DeVIC (dexamethasone, etoposide, ifosfamide and carboplatin) chemotherapy for the treatment of patients with primary central nervous system lymphoma (PCNSL). We retrospectively examined 21 patients with newly diagnosed PCNSL who received DeVIC chemotherapy followed by whole-brain radiation therapy (WBRT). The median progression-free survival (PFS) in all patients was 37.4 months and the median duration of overall survival (OS) was 47.8 months. Notably, the median duration of OS was significantly longer in patients who achieved a complete response (CR) after DeVIC chemotherapy (49.0 months) than in those without CR (12.8 months). Furthermore, we found that the overall response rate to the initial DeVIC chemotherapy was 95.2%. No treatment-related deaths were observed. Our study investigated the efficacy of DeVIC chemotherapy in PCNSL patients, and found it to result in favorable survival outcomes in these patients, thus warranting further investigation of it as a therapeutic measure against PCNSL.

The modulation of microRNAs by type I IFN through the activation of signal transducers and activators of transcription 3 in human glioma.

Type I IFNs are involved in double-stranded RNA responses. Here, we investigated the possibility that IFN-beta may induce or downregulate cellular microRNAs (miRNA) in human neoplasms and thereby use the RNA interference system to show antitumor effects. Because of its known connection to glioma biology, we focused on miR-21 among seven miRNAs influenced by IFN-beta. We analyzed the effect of IFN-beta treatment on miR-21 expression in glioma cells and intracranial glioma xenografts. IFN-beta treatment reduced miR-21 expression in glioma cells markedly, and IFN-beta administration suppressed the growth of glioma-initiating cell-derived intracranial tumors. The levels of primary miR-21 gene transcripts, precursor miR-21, and mature miR-21 decreased 6 hours after the addition of IFN-beta, indicating that the reduction in miR-21 levels was due to transcriptional suppression. We did reporter assays to elucidate the IFN-beta-mediated suppression of miR-21; the addition of signal transducers and activators of transcription 3 (STAT3)-expressing vectors induced the IFN-beta-mediated suppression of miR-21, whereas STAT3-inhibiting agents inhibited the miR-21 suppression. Thus, the results of our study show that the downregulation of miR-21 contributes to the antitumor effects of IFN-beta and that miR-21 expression is negatively regulated by STAT3 activation. These results highlight the importance of understanding the transcriptional regulation of the miRNAs involved in oncogenesis.

Synergistic induction of NY-ESO-1 antigen expression by a novel histone deacetylase inhibitor, valproic acid, with 5-aza-2'-deoxycytidine in glioma cells.

NY-ESO-1, one of the most immunogenic cancer/testis antigens, provides attractive targets for cancer immunotherapy. NY-ESO-1 has been demonstrated to be expressed in a range of solid tumors via DNA demethylation and/or histone modification; however, it has been rarely expressed in glioma. The reversibility of these epigenetic aberrations is potentially attractive for glioma treatment with DNA-methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi), leading to reactivation of silenced genes. We previously demonstrated de novo induction of NY-ESO-1 in glioma cells by DNMTi. In this study, we show that an anticonvulsant, i.e., valproic acid (VPA), also acting as an HDACi, enhances induction of NY-ESO-1 in synergy with DNMTi. Chromatin assays demonstrated that combination of DNMTi and VPA elicited significant DNA demethylation, histone H3 Lys9 demethylation, and acetylation. These findings not only shed light on an epigenetic immunotherapy, but also suggest that the silencing of NY-ESO-1 is mediated by histone modification.

Type I interferon inhibits astrocytic gliosis and promotes functional recovery after spinal cord injury by deactivation of the MEK/ERK pathway.

Formation of a glial scar is one of the major obstacles to axonal growth after injury to the adult CNS. In this study, we have addressed this issue by focusing on reactive astrocytes in a mouse model of spinal cord injury (SCI). First, we attempted to identify profile changes in the expression of astrocytic gliosis 10 days after injury by using gliosis-specific microdissection, genome-wide microarray, and MetaCore(trade mark) pathway analysis. This systematic data processing revealed many intriguing activated pathways. However, considering that proliferation/mitosis is one of the most prominent features of reactive astrocytes, we focused on the functional role of the Ras-MEK-ERK signaling cascades in reactive astrocytes. SCI-induced proliferation of reactive astrocytes in the lesion is in accordance with the increase in the expression and phosphorylation of MEK-ERK. Second, to reduce reactive gliosis after SCI, liposomes containing the interferon-beta (IFN-beta) gene were administered locally 30 min after injury. At 14 days after this treatment, GFAP-positive intensity and MEK-ERK phosphorylation at the lesion were reduced. In the animals receiving the IFN-beta gene, significant recovery of neurobehavior and parameters of electrophysiology following SCI was revealed by assessments of rotarod performance and improvements in the Basso Mouse Scale for locomotion and cortical motor-evoked potentials. SCI resulted in the degeneration of biotinylated dextran amine-labeled descending corticospinal tract axons, but the IFN-beta gene delivery induced regrowth of a large number of corticospinal tract axons. These results suggest that liposome-mediated IFN-beta gene delivery inhibits glial scar formation after SCI and promotes functional recovery.

Induction of oligodendrogenesis in glioblastoma-initiating cells by IFN-mediated activation of STAT3 signaling.

The response of cancer patients to interferon (IFN) treatment is long-lasting, indicating that IFN may act on small cancer stem cell populations. Glioma-initiating cells (GICs) can self-renew and induce the formation of heterogeneously differentiated tumor cells and are resistant to chemotherapeutic agents like temozolomide. In this study, we showed that via STAT3 signaling, IFN-beta suppressed the proliferation, self-renewal, and tumorigenesis of GICs, induced their terminal differentiation to mature oligodendroglia-like cells, and exhibited synergistic cytotoxicity with temozolomide. Therefore, IFN may be a potential therapeutic agent for inducing the terminal differentiation of GICs.