trichostatin-a and Medulloblastoma

Mutations in the gene encoding for the histone acetyltransferase (HAT) CREBBP are common driver events in multiple types of human cancer, such as small cell lung cancer (SCLC) or Sonic Hedgehog medulloblastoma (SHH MB). Therefore, therapeutic options targeting such alterations are highly desired. We used human cell lines from SCLC as well as primary mouse tumor cells and genetically engineered mouse models for SHH MB to test treatment options with histone deacetylase inhibitors (HDACi) in CREBBP wild-type and mutated tumors. In contrast to CREBBP wild-type SCLC cells, CREBBP-mutated SCLC cells showed significantly lower IC

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cell Proliferation; Cerebellar Neoplasms; CREB-Binding Protein; Disease Models, Animal; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Medulloblastoma; Mice; Mice, Transgenic; Panobinostat; Primary Cell Culture; Smoothened Receptor

Epigenetic alterations are common events in cancer. Using a genome wide methylation screen (Restriction Landmark Genomic Scanning-RLGS) we identified the gene for the dopamine receptor D4 (DRD4) as tumor-specific methylated. As DRD4 is involved in early brain development and may thus be involved in developmentally dependent tumors of the CNS in children epigenetic deregulation of DRD4 and its functional consequences were analyzed in vitro. CpG methylation of DRD4 was detected in 18/24 medulloblastomas, 23/29 ependymomas, 6/6 high-grade gliomas, 7/10 CNS PNET and 8/8 cell lines by qCOBRA and bisulfite sequencing. Real-time RT-PCR demonstrated a significantly inferior expression of DRD4 in primary tumors compared to cell lines and non-malignant control tissues. Epigenetic deregulation of DRD4 was analyzed in reexpression experiments and restoration of DRD4 was observed in medulloblastoma (MB) cells treated with 5-Aza-CdR. Reexpression was not accompanied by demethylation of the DRD4 promoter but by a significant decrease of H3K27me3 and of bound enhancer of zeste homologue 2 (EZH2). Knockdown of EZH2 demonstrated DRD4 as a direct target for inhibition by EZH2. Stimulation of reexpressed DRD4 resulted in an activation of ERK1/2. Our analyses thus disclose that DRD4 is epigenetically repressed in CNS tumors of childhood. DRD4 is a direct target of EZH2 in MB cell lines. EZH2 appears to dominate over aberrant DNA methylation in the epigenetic inhibition of DRD4, which eventually leads to inhibition of a DRD4-mediated stimulation of the ERK1/2 kinase pathway.

Topics: Apoptosis; Azacitidine; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Child; Chromatin Assembly and Disassembly; Decitabine; Dose-Response Relationship, Drug; Epigenesis, Genetic; Female; Humans; Hydroxamic Acids; Male; Medulloblastoma; Neuroectodermal Tumors, Primitive; Receptors, Dopamine D4; Sulfites; Tumor Cells, Cultured

Accumulating evidence suggests that microRNAs (miRNAs) are over- or under-expressed in tumors, and abnormalities in miRNA expression may contribute to carcinogenesis. MiR-383 was previously identified as one of the under-expressed miRNAs in medulloblastoma (MB) by miRNA expression profiling. Quantitative reverse transcription polymerase chain reaction (RT-PCR)-based miRNA assays showed an enrichment of miR-383 in normal brain. Based on these data, we speculated that miR-383 is important in MB pathogenesis. In this study, we demonstrated significant downregulation of miR-383 in 23/29 (79%) MB samples and 7/7 (100%) MB cells lines. Ectopic expression of miR-383 in MB cells led to suppression of cell growth, cell accumulation at sub-G1 phase and alteration of apoptosis-related proteins. By transcriptomic analysis and computational algorithms, we identified peroxiredoxin 3 (PRDX3) as a target gene of miR-383. Luciferase reporter assay confirmed that miR-383 negatively regulated PRDX3 by interaction between miR-383 and complementary sequences in the 3' UTR of PRDX3. MiR-383 repressed PRDX3 at transcriptional and translational levels as revealed by quantitative RT-PCR and Western blot analysis. Furthermore, depletion of PRDX3 by siRNAs resulted in similar effects as observed in miR-383-transfected cells. In conclusion, miR-383 acts as a regulator controlling cell growth of MB, at least in part, through targeting PRDX3.

Topics: Adolescent; Adult; Apoptosis; Azacitidine; Cell Cycle; Cell Line, Tumor; Cell Survival; Cerebellar Neoplasms; Child; Child, Preschool; Decitabine; Down-Regulation; Enzyme Inhibitors; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Male; Medulloblastoma; MicroRNAs; Peroxiredoxin III; RNA Interference; Young Adult

Medulloblastoma is a heterogeneous pediatric brain tumor with significant therapy-related morbidity, its five-year survival rates ranging from 30% to 70%. Improvement in diagnosis and therapy requires better understanding of medulloblastoma pathology. We used whole-genome microarray analysis to identify putative tumor suppressor genes silenced by epigenetic mechanisms in medulloblastoma. This analysis yielded 714 up-regulated genes in immortalized medulloblastoma cell line D283 on treatment with histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Dickkopf-1 (DKK1), a Wnt antagonist, was found to be up-regulated on HDAC inhibition. We examined DKK1 expression in primary medulloblastoma cells and patient samples by reverse transcriptase PCR and found it to be significantly down-regulated relative to normal cerebellum. Transfection of a DKK1 gene construct into D283 cell lines suppressed medulloblastoma tumor growth in colony focus assays by 60% (P < 0.001). In addition, adenoviral vector-mediated expression of DKK1 in medulloblastoma cells increased apoptosis fourfold (P < 0.001). These data reveal that inappropriate histone modifications might deregulate DKK1 expression in medulloblastoma tumorigenesis and block its tumor-suppressive activity.

Topics: Cell Division; Cerebellar Neoplasms; Chromatin; Colony-Forming Units Assay; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Medulloblastoma; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDIs) are a promising new class of antineoplastic agents with the ability to induce apoptosis and growth arrest of cancer cells. In addition, HDIs have been suggested to enhance the anticancer efficacy of other therapeutic regimens, such as ionizing radiation (IR) or chemotherapy. The objective of this study was to evaluate the activity of HDIs against medulloblastoma cells when applied either as single agents or in combination with IR, cytostatics, or TRAIL. The HDIs, suberoyl anilide hydroxamic acid (SAHA), sodium butyrate, and trichostatin A, were examined for their effects on the medulloblastoma cell lines, DAOY and UW228-2. We found that treatment with HDIs induced the dissipation of mitochondrial membrane potential, activation of caspase-9 and -3 and, consequently, apoptotic cell death. Moreover, all three HDIs significantly enhanced the cytotoxic effects of IR in DAOY cells. Likewise, treatment with SAHA markedly augmented the cytotoxicity of etoposide, while it had no effect on vincristine-mediated cell death. HDIs also potently increased the killing efficiency of TRAIL. TRAIL-induced, but not SAHA-induced, cell killing could be prevented by the caspase-8 inhibitor, z-IEDT-fmk. We conclude that HDIs may be useful for the treatment of medulloblastoma as monotherapy and particularly when given in combination with IR, appropriate cytostatics, or TRAIL.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Butyrates; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; DNA Fragmentation; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Etoposide; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Medulloblastoma; Membrane Glycoproteins; Membrane Potentials; Mitochondrial Membranes; Radiation-Sensitizing Agents; Radiation, Ionizing; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Vincristine; Vorinostat