trichostatin-a and Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a malignant tumour of soft tissues, occurring mainly in children and young adults. RMS cells derive from muscle cells, which due to mutations and epigenetic modifications have lost their ability to differentiate. Epigenetic modifications regulate expression of genes responsible for cell proliferation, maturation, differentiation and apoptosis. HDAC inhibitors suppress histone acetylation; therefore, they are a promising tool used in cancer therapy. Trichostatin A (TsA) is a pan-inhibitor of HDAC. In our study, we investigated the effect of TsA on RMS cell biology. Our findings strongly suggest that TsA inhibits RMS cell proliferation, induces cell apoptosis, and reactivates tumour cell differentiation. TsA up-regulates miR-27b expression, which is involved in the process of myogenesis. Moreover, TsA increases susceptibility of RMS cells to routinely used chemotherapeutics. In conclusion, TsA exhibits anti-cancer properties, triggers differentiation, and thereby can complement an existing spectrum of chemotherapeutics used in RMS therapy.

Topics: Acetylation; Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Epigenesis, Genetic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MicroRNAs; Muscle Development; Rhabdomyosarcoma

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1 Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors.

Topics: Cell Line, Tumor; Chromosomal Instability; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Histone Deacetylases; Humans; Hydroxamic Acids; Mutation; Phosphoric Diester Hydrolases; Rhabdomyosarcoma; RNA, Small Interfering; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; TATA-Binding Protein Associated Factors; Valproic Acid

Rhabdomyosarcoma (RMS), which can be classified as embryonal RMS (ERMS) and alveolar RMS (ARMS), represents the most frequent soft tissue sarcoma in the pediatric population; the latter shows greater aggressiveness and metastatic potential with respect to the former. Epigenetic alterations in cancer include DNA methylation changes and histone modifications that influence overall gene expression patterns. Different tumor subtypes are characterized by distinct methylation signatures that could facilitate early disease detection and greater prognostic accuracy.. A genome-wide approach was used to examine methylation patterns associated with different prognoses, and DNA methylome analysis was carried out using the Agilent Human DNA Methylation platform. The results were validated using bisulfite sequencing and 5-aza-2'deoxycytidine treatment in RMS cell lines. Some in vitro functional studies were also performed to explore the involvement of a target gene in RMS tumor cells.. In accordance with the Intergroup Rhabdomyosarcoma Study (IRS) grouping, study results showed that distinct methylation patterns distinguish RMS subgroups and that a cluster of protocadherin genes are hypermethylated in metastatic RMS. Among these, PCDHA4, whose expression was decreased by DNA methylation, emerged as a down-regulated gene in the metastatic samples. As PCDHA4-silenced cells have a significantly higher cell proliferation rate paralleled by higher cell invasiveness, PCDHA4 seems to behave as a tumor suppressor in metastatic RMS.. Study results demonstrated that DNA methylation patterns distinguish between metastatic and non-metastatic RMS and suggest that epigenetic regulation of specific genes could represent a novel therapeutic target that could enhance the efficiency of RMS treatments.

Topics: Azacitidine; Biopsy; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Cluster Analysis; Cytidine Triphosphate; DNA Methylation; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Hydroxamic Acids; Neoplasm Metastasis; Neuropeptides; Promoter Regions, Genetic; Protocadherins; Receptors, Cell Surface; Rhabdomyosarcoma; Transcriptome

Rhabdomyosarcomas (RMS) are highly malignant pediatric sarcomas. We have discovered that the gene encoding the major histocompatibilty complex class II transactivator, CIITA, is silenced in cells representing both major subtypes of RMS. Silencing of CIITA prevents the IFN-γ inducible expression of MHC class II genes in these cells. Overexpression of CIITA in these cells can restore MHC expression. We have found that IFN-γ signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells but does show a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have found that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-γ treatment, is blocked in both types of RMS cells. In RD cells, treatment with a histone deacetylase inhibitor (TSA) reverses the silencing of CIITA. In SJRH30 cells, treatment with DNA methyltransferase inhibitors and TSA cooperatively restores CIITA expression. Surprisingly, we have also shown that the expression of two components of the immunoproteasome, which are embedded in the class II locus, is stimulated by IFN-γ in certain RMS cells in the absence of stimulation by CIITA. CIITA overexpression can also activate the expression of these genes, indicating that the immunoproteasome genes LMP2 and LMP7 can be activated by both CIITA dependent and CIITA independent pathways.

Topics: Acetylation; Azacitidine; Cell Line, Tumor; Chromatin Immunoprecipitation; Decitabine; Epigenesis, Genetic; Flow Cytometry; Gene Expression Profiling; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Interferon-gamma; Nuclear Proteins; Promoter Regions, Genetic; Rhabdomyosarcoma; Signal Transduction; Trans-Activators

The active copy of the imprinted gene H19 is turned off by inappropriate methylation in several pediatric tumors including Wilms' Tumour and embryonal rhabdomyosarcoma. H19 controls in cis the linked Insulin-like Growth Factor 2 (IGF2) gene, encoding an important growth factor. Recent work has suggested that methylation of a gene may lead to deacetylation of its associated histones and that silenced genes can be reactivated by increasing histone acetylation levels.. Treatment of a rhabdomyosarcoma cell line which has a silent, methylated H19 gene with histone deacetylase (HDAC) inhibitors under conditions which gave maximal hyperacetylation of histone 4, both globally and at the H19 gene itself could not reactivate H19 or affect the active Insulin-like Growth Factor 2 (IGF2) gene, but caused clear up-regulation of the Tissue-type Plasminogen Activator (TPA) gene, a non-imprinted gene known to respond to changes in histone acetylation. In contrast, mild treatment of the cells with the methylation inhibitor 5-AzaC-2'-deoxycytidine (AzaC) on its own was able to reactivate H19. Combining AzaC treatment with HDAC inhibitors gave a reduced rather than enhanced reactivation. These findings were confirmed in mouse primary liver and kidney explants which maintain normal imprinting, where we also found that the silent Igf2 gene could not be reactivated by HDAC inhibitors.. These results suggest that DNA methylation rather than histone acetylation is the primary determinant of silencing of H19 in rhabdomyosarcoma.

Topics: Acetylation; Animals; Azacitidine; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Cells, Cultured; DNA Methylation; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Insulin-Like Growth Factor II; Kidney; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pregnancy; Rhabdomyosarcoma; RNA; RNA, Long Noncoding; RNA, Untranslated; Tissue Plasminogen Activator

There is a strong correlation between the acetylation status of nucleosomal histones and transcriptional activity. Here we show that the histone deacetylase inhibitor trichostatin A (TSA) activates reporter gene constructs driven by the human platelet-derived growth factor B (PDGF-B) gene promoter. This activation showed an inverse correlation with the cell type-specific transcriptional activities of the promoter. The TSA response was minimal in three tumor cell lines that exhibit high-level promoter activity. In JEG-3 choriocarcinoma cells, however, where the basal promoter activity is considerably lower, there was a strong response to TSA. This was in contrast to constructs that included a PDGF-B enhancer, which were refractory to TSA effects, indicating a possible function of the enhancer in modulating acetylation status. Analysis of PDGF-B promoter mutants with respect to TSA induction revealed no specific TSA-responsive element, but suggested that association of nonacetylated histones to the PDGF-B promoter may be a default process in the absence of enhancer activation. TSA treatment of JEG-3 cells, either alone or in combination with the demethylating agent 5-azacytidine, failed to activate the silenced endogenous PDGF-B transcript, however, which appears to be repressed by additional mechanisms.

Topics: Adenocarcinoma; Breast Neoplasms; Carcinoma, Hepatocellular; Choriocarcinoma; Chromosomes; DNA Methylation; Enhancer Elements, Genetic; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Introns; Liver Neoplasms; Mutagenesis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-sis; Rhabdomyosarcoma; Transcription, Genetic; Tumor Cells, Cultured