Rossella Rota

Down-regulation of RNA Editing in Pediatric Astrocytomas ADAR2 EDITING ACTIVITY INHIBITS CELL MIGRATION AND PROLIFERATION

Since alterations in post-transcriptional events can contribute to the appearance and/or progression of cancer, we investigated whether RNA editing, catalyzed by the ADAR (adenosine deaminases that act on RNA) enzymes, is altered in pediatric astrocytomas. We find a decrease in ADAR2 editing activity that seems to correlate with the grade of malignancy in children. Despite the loss of ADAR2 editing activity in tumor tissues, the high grade astrocytomas do not exhibit alterations in ADAR2 expression when compared with their specific control tissues. However, high expression levels of ADAR1 and ADAR3 were found in tumors when compared with normal tissues dissected in the same area of the brain. We reintroduced either ADAR2 or the inactive version of ADAR2 in three astrocytoma cell lines (U118, A172, U87). The “reverted” editing status is necessary and sufficient for a significant decrease in cell malignant behavior as measured by proliferation, cell cycle, and migration assays. We show that elevated levels of ADAR1, as found in astrocytomas, do indeed interfere with ADAR2 specific editing activity. Furthermore, we show that the endogenous ADAR1 can form heterodimers with ADAR2 in astrocytes.

Deregulated expression of miR-26a and Ezh2 in rhabdomyosarcoma.

172 Cell Cycle 2009; Vol. 8 Issue 1 Rhabdomyosarcoma (RMS) is a highly malignant pediatric tumor that is thought to derive from mesenchymal cells already committed to become skeletal muscle cells.1 The majority of RMS tumors continues to express high levels of early inducers of muscle differentiation, such as MyoD and myogenin, indicating that aberrant differentiation pathways may participate in rhabdomyosarcoma tumorigenesis.2 Pediatric RMS is classified into two major types, embryonal RMS (ERMS) and the more aggressive alveolar RMS (ARMS). The embryonal and alveolar types of RMS differ in histology, genetic markers, current treatment protocols and prognosis. In particular, most alveolar rhabdomyosarcomas contain either the common t(2;13)(q35;q14) or the rare t(1;13)(p36;q14) translocations that form respectively PAX3-FKHR and PAX7-FKHR fusion proteins.1 Since RMS is a highly invasive malignant neoplasm, its complete surgical resection is often difficult. Therefore most of young patients require a multimodal therapy that is associated with significant acute toxicity and long-term side effects. These clinical drawbacks raise the necessity of alternative therapies tailored towards new molecular targets. Recently, the strong relationship between microRNA (miRNA) deregulated expression and cancer became evident also in human sarcomas.3 miRNAs are short non-coding RNA molecules able to regulate gene expression, affecting the stability and/or translation of target mRNAs.4 They have been established to have an important role in development and lineage differentiation, including that of the skeletal muscle system.4 A growing number of reports confirmed that clustering of the samples on the basis of their miRNA expression might be helpful for determining the developmental origin of tissues and even the cancer diagnosis.5 Numerous data suggest that each tumor type is characterized by a specific miRNA expression profile, and sometimes a given miRNA can be upregulated in some tumors and downregulated in others.6 Moreover, differences in miRNA profiles also exist among various histopathological variants of the same tumor type.7 To investigate whether miRNAs are differentially expressed in human rhabdomyosarcoma we analyzed the levels of a selected group of 20 miRNAs among those already shown to be involved in skeletal muscle differentiation and potentially in cancerogenesis (Table 1). Using RealTime polymerase chain reaction technology, we measured the expression of mature miRNA molecules in two human rhabdomyosarcoma cell lines (Table 1), embryonal RD and alveolar Rh-30, the latter expressing the PAX3-FKHR fusion protein. As rhabdomyosarcoma is believed to derive from skeletal muscle progenitor cells, we compared miRNA expression in RD and Rh-30 with that of normal human proliferating myoblasts cultured in growth medium (Skeletal Muscle Cells, Promo Cell, Germany) SkMC GM (Table 1). In RD cells eight miRNAs (40.0%) were upregulated and two (10.0%) were downregulated, compared to SkMC GM with more than a 2-fold change. No significant difference was observed for the remaining 10 miRNAs (50.0%) analyzed. Conversely, in Rh-30 cells, only one miRNA (5.0%) was upregulated whereas twelve (60.0%) were downregulated at different degrees, and seven (35.0%) remained unchanged (Table 1). These results show a pronounced downregulation of the expression of several miRNAs in the Rh-30 cell line compared to SkMC GM control cells. This is in accordance with the aggressive character of Rh-30 cells, and with the observation that a lower expression of miRNAs has been reported in tumors than in their normal tissue counterparts.4,5,8 Consistently, an overall lower expression of miRNAs has been clinically related to a more differentiated and aggressive phenotype in hepatocellular carcinomas, where mature miRNA expression level has been found to be the lowest, and patient survival the poorer.9 A substantial difference in microRNA expression between the two cell lines is also evident. The expression of 19 out of 20 miRNAs (95.0%) in RD is significantly higher than in Rh-30 (RD/Rh-30 column in Table 1), supporting the assumption that RD cells are characterized by a more differentiated phenotype. This has been already supported by the poor expression of Pax3, a known inhibitor of muscle differentiation that, conversely, exerts transcriptional activity in Rh-30 under the form of the PAX3-FKHR fusion protein.10 To evaluate the modulation of miRNAs in a human skeletal muscle differentiation model, we treated the SkMC myoblasts for 10 days with a differentiating medium (PromoCell).11 Under these conditions more than 70% SkMC converted into multinucleate myotubes (SkMC DM). Results on miRNA expression revealed a general upregulation of the miRNAs analyzed in differentiating SkMC DM compared to SkMC GM (Table 1). Particularly, miR-1 and miR-26a, two major inducers of myogenesis, are strongly expressed among those involved in muscle differentiation (Table 1). Interestingly, they appeared to be the most upregulated of this group of miRNAs also in RD cells, strengthening the assumption that RD cells could be arrested at a later phase of differentiation compared to Rh-30 cells.10 Among miRNAs previously involved in other types of cancer, miR17-5p, miR-19a, miR-19b miR-20 and miR-106a showed a coordinated expression in each cell line, as expected, since they belong to the two homologous clusters, miR-17-92 and miR-106a.12 They were not consistently modulated, being modestly upregulated in RD and SkMC DM and downregulated in Rh-30 (Table 1). Recently, beyond a wellknown oncogenic role in several human cancers,7 the miR-17-92 cluster has been reported to have tumor-suppressive features, representing a context-dependent miRNA.13 miR-221 and miR-222, generally well expressed in other human tumors compared to normal tissue,7 were downregulated in both tumor cell lines (Table 1). In a recent report on primary rhabdomyosarcomas these miRNAs have been shown to be not upregulated.4 To spotlight the clinical relevance of the miRNAs measured in RD and Rh-30 cell lines, we performed a Real Time PCR analysis of selected miRNAs (bolded in Table 1) on a group of five rhab*Correspondence to: Rossella Rota; Laboratory of Angiogenesis; Ospedale Pediatrico Bambino Gesù; Research Institute; Piazza S. Onofrio 4; Roma 00165 Italy; Tel.: 39.06.6859.2648; Fax: 39.06.6859.2904; Email: rota@opbg.net/Antonio Giordano; Sbarro Institute for Cancer Research and Molecular Medicine; Center for Biotechnology; College of Science and Technology; Temple University; BioLife Science Bldg. Ste. 333; 1900 North 12th Street; Philadelphia; PA 19122 USA; Tel.: 2152049520; Fax 2152049522; email: giordano@temple.edu

Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer

In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti‐VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti‐VEGF therapy in a different animal model which closely mimics human ROP.

Helper-dependent adenovirus for the gene therapy of proliferative retinopathies: stable gene transfer, regulated gene expression and therapeutic efficacy

Ocular neovascular disorders, such as diabetic retinopathy and age‐related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper‐dependent adenovirus (HD‐Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization.

Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21 Cip1 expression and hampers tumour cell growth in vitro and in vivo

Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21Cip1 level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx)

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation

Inhibitor of DNA-binding (Id) proteins prevent cell differentiation, promote growth and sustain tumour development. They do so by binding to E proteins and other transcription factors through the helix-loop-helix (HLH) domain, and inhibiting transcription. This makes HLH-mediated Id protein interactions an appealing therapeutic target. We have used the dominant interfering HLH dimerization mutant 13I to model the impact of Id inhibition in two human neuroblastoma cell lines: LA-N-5, similar to immature neuroblasts, and SH-EP, resembling more immature precursor cells. We have validated 13I as an Id inhibitor by showing that it selectively binds to Ids, impairs complex formation with RB, and relieves repression of E protein-activated transcription. Id inactivation by 13I enhances LA-N-5 neural features and causes SH-EP cells to acquire neuronal morphology, express neuronal proteins such as N-CAM and NF-160, proliferate more slowly, and become responsive to retinoic acid. Concomitantly, 13I augments the cell-cycle inhibitor p27Kip1 and reduces the angiogenic factor vascular endothelial growth factor. These effects are Id specific, being counteracted by Id overexpression. Furthermore, 13I strongly impairs tumorigenic properties in agar colony formation and cell invasion assays. Targeting Id dimerization may therefore be effective for triggering differentiation and restraining neuroblastoma cell tumorigenicity.

EZH2 Down-Regulation Exacerbates Lipid Accumulation and Inflammation in in Vitro and in Vivo NAFLD

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent, chronic liver diseases, worldwide. It is a multifactorial disease caused by complex interactions between genetic, epigenetic and environmental factors. Recently, several microRNAs, some of which epigenetically regulated, have been found to be up- and/or down-regulated during NAFLD development. However, in NAFLD, the essential role of the Polycomb Group protein Enhancer of Zeste Homolog 2 (EZH2), which controls the epigenetic silencing of specific genes and/or microRNAs by trimethylating Lys27 on histone H3, still remains unknown. In this study, we demonstrate that the nuclear expression/activity of the EZH2 protein is down-regulated both in livers from NAFLD rats and in the free fatty acid-treated HepG2. The drop in EZH2 is inversely correlated with: (i) lipid accumulation; (ii) the expression of pro-inflammatory markers including TNF-α and TGF-β; and (iii) the expression of miR-200b and miR-155. Consistently, the pharmacological inhibition of EZH2 by 3-Deazaneplanocin A (DZNep) significantly reduces EZH2 expression/activity, while it increases lipid accumulation, inflammatory molecules and microRNAs. In conclusion, the results of this study suggest that the defective activity of EZH2 can enhance the NAFLD development by favouring steatosis and the de-repression of the inflammatory genes and that of specific microRNAs.

Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.

BackgroundEmbryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.MethodsEmbryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.ResultsHere we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.ConclusionsThese results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

Enhancer of zeste homolog 2 (EZH2) in pediatric soft tissue sarcomas: first implications

Soft tissue sarcomas of childhood are a group of heterogeneous tumors thought to be derived from mesenchymal stem cells. Surgical resection is effective only in about 50% of cases and resistance to conventional chemotherapy is often responsible for treatment failure. Therefore, investigations on novel therapeutic targets are of fundamental importance. Deregulation of epigenetic mechanisms underlying chromatin modifications during stem cell differentiation has been suggested to contribute to soft tissue sarcoma pathogenesis. One of the main elements in this scenario is enhancer of zeste homolog 2 (EZH2), a methyltransferase belonging to the Polycomb group proteins. EZH2 catalyzes histone H3 methylation on gene promoters, thus repressing genes that induce stem cell differentiation to maintain an embryonic stem cell signature. EZH2 deregulated expression/function in soft tissue sarcomas has been recently reported. In this review, an overview of the recently reported functions of EZH2 in soft tissue sarcomas is given and the hypothesis that its expression might be involved in soft tissue sarcomagenesis is discussed. Finally, the therapeutic potential of epigenetic therapies modulating EZH2-mediated gene repression is considered.