HDAC Inhibition for the Treatment of Epithelioid Sarcoma: Novel Cross Talk Between Epigenetic Components.

UNLABELLED: Epithelioid sarcoma is a rare neoplasm uniquely comprised of cells exhibiting both mesenchymal and epithelial features. Having propensity for local and distant recurrence, it poses a diagnostic dilemma secondary to pathologic complexity. Patients have dismal prognosis due to lack of effective therapy. HDAC inhibitors (HDACi) exhibit marked antitumor effects in various malignancies. The studies here demonstrate that pan-HDAC inhibitors constitute novel therapeutics versus epithelioid sarcoma. Human ES cells (VAESBJ, HS-ES, Epi-544) were studied in preclinical models to evaluate HDACi effects. Immunoblot and RT-PCR were used to evaluate expression of acetylated tubulin, histones H3/H4, EZH2 upon HDACi. MTS and clonogenic assays were used to assess the impact of HDACi on cell growth. Cell culture assays were used to evaluate the impact of HDACi and EZH2-specific siRNA inhibition on cell-cycle progression and survival. Unbiased gene array analysis was used to identify the impact of HDACi on epithelioid sarcoma gene expression. Xenografts were used to evaluate epithelioid sarcoma tumor growth in response to HDACi. HDAC inhibition increased target protein acetylation and abrogated cell growth and colony formation in epithelioid sarcoma cells. HDACi induced G(2) cell-cycle arrest and marked apoptosis, and reduced tumor growth in xenograft models. HDACi induced widespread gene expression changes, and EZH2 was significantly downregulated. EZH2 knockdown resulted in abrogated cell growth in vitro. IMPLICATIONS: The current study suggests a clinical role for HDACi in human epithelioid sarcoma, which, when combined with EZH2 inhibitors, could serve as a novel therapeutic strategy for epithelioid sarcoma patients. Future investigations targeting specific HDAC isoforms along with EZH2 may potentially maximizing treatment efficacy.

PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway.

PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 µM) and analyzed for cell proliferation by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway.

Co-expression network analysis of differentially expressed genes associated with metastasis in prolactin pituitary tumors.

The aim of the present study was to construct a co­expression network of differently expressed genes (DEGs) in prolactin pituitary (PRL) tumor metastasis. The gene expression profile, GSE22812 was downloaded from the Gene Expression Omnibus database, and including five non­invasive, two invasive and six aggressive­invasive PRL tumor samples. Compared with non­invasive samples, DEGs were identified in invasive and aggressive­invasive samples using a limma package in R language. The expression values of DEGs were hierarchically clustered. Next, Gene Ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis of DEGs were performed via The Database for Annotation, Visualization and Integrated Discovery. Finally, gene pairs of DEGs between non­invasive and aggressive­invasive samples were identified using the Spearman cor( ) function in R language. Compared with the non­invasive samples, 61 and 89 DEGs were obtained from invasive and aggressive­invasive samples, respectively. Cluster analysis showed that four genes were shared by the two samples, including upregulated solute carrier family 2, facilitated glucose transporter member 11 (SLC2A11) and teneurin transmembrane protein 1 (TENM1) and downregulated importin 7 (IPO7) and chromogranin B (CHGB). In the invasive samples, the most significant GO terms responded to cyclic adenosine monophosphate and a glucocorticoid stimulus. However, this occurred in the cell cycle, and was in response to hormone stimulation in aggressive­invasive samples. The co­expression network of DEGs showed different gene pairs and modules, and SLC2A11 and CHGB occurred in two co­expression networks within different co­expressed pairs. In the present study, the co­expression network was constructed using bioinformatics methods. SLC2A11, TENM1, IPO7 and CHGB are hypothesized to be closely associated with metastasis of PRL. Furthermore, CHGB and SLC2A11 may be significant in PRL tumor progression and serve as molecular biomarkers for PRL tumors. However, further investigation is required to confirm the current results.

mGlu3 receptor blockade inhibits proliferation and promotes astrocytic phenotype in glioma stem cells.

We have characterised, using both in vivo and in vitro methods, the effects of the metabotropic glutamate receptor subtype 3 (mGlu3) antagonist (LY341495) and agonist (LY379268) on the proliferation and differentiation of glioma stem cells (GSC). For in vitro studies, a CCK-8 assay was used to determine the cell proliferation, flow cytometry was performed to determine cell cycle phases, and immunohistochemistry and laser confocal microscopy were employed to detect CD133 expression. For in vivo studies, GSCs were injected into nude mice treated with either LY379268 or LY341495 and the growth of the tumours was measured after 3 weeks. When compared with controls, the proliferation rates and proportion of cells in S phase within the LY341495 treated group decreased in a time-dependent manner. In the presence of differentiation medium containing LY341495, GSC differentiation was diverted into an astrocyte rather than neuronal phenotype. The growth rate and volume of tumours injected into nude mice was reduced in LY341495 treated mice compared with controls. Thus pharmacological blockade of mGlu3 receptor signalling pathway significantly inhibits the growth and proliferation of GSCs both in vitro and in vivo while promoting differentiation to astrocytes. These results further implicate mGlu3 in the biology of glioma and as a target for continued research.

A novel TP53 somatic mutation involved in the pathogenesis of pediatric choroid plexus carcinoma.

BACKGROUND: Choroid plexus carcinoma (CPC) is an uncommon, aggressive, malignant, central nervous system neoplasm that typically occurs in children, presenting with the signs and symptoms of intracranial hypertension and cerebrospinal fluid obstruction. CASE REPORT: We report the case of a 2.5-year-old girl with CPC. The tumor was subtotally removed by microsurgery, followed by gamma knife radiosurgery for the residual lesion. H&E staining indicated that this was a rare case of CPC. Neuropathological studies, assayed by immunohistochemical staining, showed that the tumor sample was positive to antibodies against S-100, CgA, AE1/AE3 (cytokeratin), Ki-67, INI1 and TP53, and was negative to antibodies against Nestin, GFAP, CD133, EMA and AFP. Moreover, stainings for transthyretin and vimentin were focally positive. Interestingly, direct DNA sequencing of the paraffin-embedded tumor sample identified a novel R248Q mutation in the TP53 gene. In contrast to previous reports suggesting that TP53 germline mutations were associated with the pathogenesis of CPC, here we provide a rare case of CPC with TP53 somatic mutation, as evidence that the peritumoral tissue possesses the non-mutant TP53 allele. CONCLUSIONS: Our finding suggests that TP53 somatic mutations, in addition to its germline mutations, may also be involved in the pathogenesis of pediatric CPC.

MiR-155 is a liposarcoma oncogene that targets casein kinase-1α and enhances ß-catenin signaling.

Liposarcoma can be an aggressive, debilitating, and fatal malignancy. In this study, we identifed miRNAs associated with the differentiation status of liposarcoma to gain insight into the basis for its progression. miRNA expression profiles determined in human tumors and normal fat specimens identified a dedifferentiated tumor expression signature consisting of 35 miRNAs. Deregulated miRNA expression was confirmed in a second independent sample cohort. The miR-155 was the most overexpressed miRNA and functional investigations assigned an important role in the growth of dedifferentiated liposarcoma cell lines. Transient or stable knockdown of miR-155 retarded tumor cell growth, decreased colony formation, and induced G(1)-S cell-cycle arrest in vitro and blocked tumor growth in murine xenografts in vivo. We identified casein kinase 1α (CK1α) as a direct target of miR-155 control which enhanced ß-catenin signaling and cyclin D1 expression, promoting tumor cell growth. In summary, our results point to important functions for miR-155 and ß-catenin signaling in progression of liposarcoma, revealing mechanistic vulnerabilities that might be exploited for both prognostic and therapeutic purposes.

Expression and cellular distribution of vascular endothelial growth factor-C system in cortical tubers of the tuberous sclerosis complex.

Cortical tubers are malformations of cortical development in patients with tuberous sclerosis complex (TSC), and highly associated with pediatric intractable epilepsy. Recent evidence has shown that signaling mediated through vascular endothelial growth factor-C (VEGF-C) and its receptors, VEGFR-2 and VEGFR-3, has direct effects on both neurons and glial cells. To understand the potential role of VEGF-C system in the pathogenesis of cortical tubers, we investigated the expression patterns of VEGF-C signaling in cortical tubers compared with age-matched normal control cortex (CTX). We found that VEGF-C, VEGFR-2 and VEGFR-3 were clearly upregulated in tubers at both the mRNA and protein levels, compared with CTX. The in situ hybridization and immunostaining results demonstrated that VEGF-C, VEGFR-2 and VEGFR-3 were highly expressed in dysplastic neurons (DNs), giant cells (GCs) and reactive astrocytes within tubers. Most DNs/GCs expressing VEGF-C and its receptors co-labeled with neuronal rather than astrocytic markers, suggesting a neuronal lineage. In addition, protein levels of Akt-1, p-Bad and ERK1/2, the important downstream factors of the VEGF-C pathway, were significantly increased in cortical tubers, indicating involvement of VEGF-C-dependent prosurvival signaling in cortical tubers. Taken together, our results suggest a putative role for the VEGF-C signaling pathway in the pathogenesis of cortical tubers.

In nonfunctional pituitary adenomas, estrogen receptors and slug contribute to development of invasiveness.

CONTEXT: Clinical studies suggest that an imbalance in the actions of estrogen receptor (ER)-α and ERß is associated with invasion of tumors of epithelial origin. Both ER have been detected in the pituitary adenomas (PA). Our previous study together with other reports suggests that an imbalance between ERα and ERß contributes to the pathogenesis and biological behavior of PA including invasion. However, the roles of the two ER in invasiveness of PA have not been clarified. OBJECTIVE: The expression of ER, Slug, and E-cadherin in 41 cases of nonfunctional PA (NFPA) were determined to evaluate whether ER were related to the invasiveness of NFPA. Furthermore, we aimed by analysis of the correlation between ER and E-cadherin or Slug to understand molecular mechanisms related to invasiveness. METHODS: Immunohistochemistry, RT-PCR, and Western blot were performed. RESULTS: Nuclear ERα staining was significantly stronger in invasive NFPA than noninvasive ones (P < 0.01). In contrast, nuclear ERß staining was significantly weaker in invasive NFPA than in noninvasive ones (P < 0.01). Both E-cadherin mRNA and protein were decreased in invasive NFPA compared with noninvasive ones. Moreover, Slug, a repressor of E-cadherin, was significantly increased in invasive over noninvasive NFPA (P < 0.01). There were significant correlations between ER and Slug or E-cadherin in NFPA, in which Slug was positively correlated with ERα and inversely correlated with ERß, whereas E-cadherin was positively correlated with ERß and inversely correlated with ERα. CONCLUSIONS: ERα and ERß may act in opposite directions to regulate the Slug-E-cadherin pathway and to affect invasiveness of NFPA.

The modulatory effects of bHLH transcription factors with the Wnt/beta-catenin pathway on differentiation of neural progenitor cells derived from neonatal mouse anterior subventricular zone.

The subventricular zone (SVZ) located adjacent to the lateral ventricles is the major site where neural progenitor cells (NPCs) are concentrated in the adult brain. NPCs in the anterior subventricular zone (SVZa) generate neuronal precursors and migrate along a highly localized pathway--the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into interneurons. To investigate the modulatory effects of basic helix-loop-helix (bHLH) transcription factors on differentiation from SVZa NPCs, we firstly examined the distribution of bHLH family members (Mash1, Id2, and Hes1) in cultured mouse SVZa NPCs and evaluated their regulatory effects on differentiation by transfection with Mash1, Id2, or Hes1 eukaryotic expression plasmid. Furthermore, we assessed the effects of bHLH transcription factors on the expression of downstream molecules of the Wnt/beta-catenin pathway, beta-catenin and (Glycogen synthase kinase-3beta). Our results demonstrated that Mash1, Id2, Hes1 were all widely expressed in in vitro progenies from mouse SVZa NPCs. Analyses of SVZa NPCs transfected with eukaryotic expression plasmids showed that Mash1 promoted neuronal differentiation from SVZa NPCs, while Id2 and Hes1 repressed neuronal differentiation. In addition, we found that Id2 and Hes1 simulated expression of beta-catenin and GSK-3beta, while Mash1 inhibited their expression. Our results suggest that the classic bHLH transcription factors, Mash1, Id2 and Hes1, play important roles in the regulation of differentiation from SVZa NPCs. This modulation is possibly mediated by a coordination of bHLH and Wnt/beta-catenin signaling.

SVZa neural stem cells differentiate into distinct lineages in response to BMP4.

Neural stem cells (NSCs) reside in the anterior portion of the forebrain subventricular zone (SVZa) and generate the progenitors which will differentiate into neurons, and via a tangential migratory pathway, known as the rostral migratory stream (RMS), migrate to the olfactory bulbs (OB). Bone morphogenetic proteins (BMPs) play significant roles in neural development at different stages and locations, but their roles have not been determined in the SVZa. To explore possible roles of BMPs in SVZa NSCs, BMP4 at various concentrations were tested for their capacity to induce SVZa NSCs. The expression of BMP4 was also examined in living cells using a reportor vector, in which the BMP4 promotor was conjugated with red fluorescent protein (RFP). In the meantime, the differentiation of SVZa NSCs was dynamically monitored by using reportor vectors of the Nestin enhancer and the promoters of TH and GFAP. In the OB, high expression of BMP4 was found using both promoter activity analysis and in situ hybridization. However, low BMP4 expression was found in the RMS and only moderate expression of BMP4 was displayed in the SVZa. The results also demonstrated that low concentrations (1-5 ng/ml) of BMP4 promoted the proliferation of SVZa NSCs but high concentrations (10-100 ng/ml) of BMP4 inhibited this proliferation. BMP4 enhanced neuron commitment before 4 days but inhibited it after 4 days. As the antagonist of BMP4, Noggin almost completely blocked all these BMP4 responses. Thus, our findings indicate that BMP4 promotes the exit from the cell cycle and triggers the differentiation of neuron progenitors in the OB. BMP4 also promotes the proliferation of the committed neuron progenitors in the RMS, but in the SVZa, BMP4 may facilitate the commitment of NSCs into astrocytes.