Federica Verginelli

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

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.

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.

Transcription factors FOXG1 and Groucho/TLE promote glioblastoma growth

Glioblastoma (GBM) is the most common and deadly malignant brain cancer, with a median survival of less than two years. GBM displays a cellular complexity that includes brain tumour-initiating cells (BTICs), which are considered as potential key targets for GBM therapies. Here we show that the transcription factors FOXG1 and Groucho/TLE are expressed in poorly differentiated astroglial cells in human GBM specimens and in primary cultures of GBM-derived BTICs, where they form a complex. FOXG1 knockdown in BTICs causes downregulation of neural stem/progenitor and proliferation markers, increased replicative senescence, upregulation of astroglial differentiation genes, and decreased BTIC-initiated tumour growth upon intracranial transplantation into host mice. These effects are phenocopied by Groucho/TLE knockdown or dominant-inhibition of the FOXG1:Groucho/TLE complex. These results provide evidence that transcriptional programs regulated by FOXG1 and Groucho/TLE are important for BTIC-initiated brain tumour growth, implicating FOXG1 and Groucho/TLE in GBM tumorigenesis.

Transcription factor Runx1 inhibits proliferation and promotes developmental maturation in a selected population of inner olfactory nerve layer olfactory ensheathing cells

The olfactory system undergoes persistent regeneration throughout life. Olfactory ensheathing cells (OECs) are a specialized class of glia found exclusively in the olfactory system. OECs wrap olfactory sensory neuron axons and support their growth from the olfactory epithelium, and targeting to the olfactory bulb, during development and life-long regeneration. Because of this function and their ability to cross the boundary between central and peripheral nervous systems, OECs are attractive candidates for cell-based regenerative therapies to promote axonal repair in the injured nervous system. OECs are a molecularly, topologically and functionally heterogeneous group of cells and the mechanisms underlying the development and function of specific OEC subpopulations are poorly defined. This situation has affected the outcome and interpretation of OEC-based regenerative strategies. Here we show that the transcription factor Runx1 is selectively expressed in OECs of the inner olfactory nerve layer of the mouse olfactory bulb and in their precursors in the OEC migratory mass. Furthermore, we provide evidence that in vivo knockdown of mouse Runx1 increases the proliferation of the OECs in which Runx1 is expressed. Conversely, Runx1 overexpression in primary cultures of OECs reduces cell proliferation in vitro. Decreased Runx1 activity also leads to an increase in Runx1-expressing OEC precursors, with a parallel decrease in the number of more developmentally mature OECs. These results identify Runx1 as a useful new marker of a distinct OEC subpopulation and suggest that Runx1 is important for the development of this group of OECs. These observations provide an avenue for further exploration into the molecular mechanisms underlying the development and function of specific OEC subpopulations.

Hyper-activation of Notch3 amplifies the proliferative potential of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

Abstract 3417: Ezh2 is up-regulated and correlates with Ki67 and CD31 expression in human pediatric rhabdomyosarcoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Rhabdomyosarcoma (RMS) is a pediatric tumor believed to originate from perturbations of the normal development of the muscle lineage. The Polycomb Group (PcG) protein Ezh2, which was originally implicated in inhibiting homeobox gene expression during embryonic development, was recently shown to act as a negative regulator of muscle differentiation. Moreover, Ezh2 is a histone methyltransferase that is aberrantly overexpressed in most human cancers. Comparing the level of the expression of Ezh2 in RMS cell lines, a small group of tumor samples with normal skeletal muscle myoblasts (SkMC), and normal muscle tissues, we have previously observed that Ezh2 is highly upregulated in rhabdomyosarcoma tumors (Ciarapica et al., 2009). The aim of this study was to characterize the relevance of Ezh2 expression in RMS. Ezh2 expression was analyzed by immunohistochemistry in a cohort of 32 RMS primary tumors and correlated with the expression of the cell proliferation marker, Ki67, and the angiogenic marker, CD31. Results of these expression studies were then correlated with the clinicopathological features of the tumor samples. No significant difference in the clinicopathological variables was observed between the two major histophatological groups of RMS, the most aggressive alveolar RMS (ARMS) and the embryonal RMS (ERMS). Ezh2 expression was not correlated with the clinical parameters of the cohorts, but strikingly, a significant positive correlation was observed between Ezh2 and Ki67 expression in the whole RMS sample (r0 = 0,560; p = 0,013). The number of the large vessels was correlated with the total number of vessels (r0= 0,730; p<0,0001) and with the expression of Ki67 (r0= 0,421; p=0,02). In ARMS samples, the number of large vessels was significantly correlated with the expression of Ezh2 (r0= 0,559; p=0,047), the total number of vessels (r0= 0,781; p=0,02), and the expression of Ki67 (r0= 0,738; p=0,004). This study confirmed our previous observation regarding the aberrantly high expression of Ezh2 in rhabdomyosarcoma tumors on a larger cohort of samples. Moreover, the present data established a positive correlation between Ezh2 expression and the proliferative and angiogenic potential of rhabdomyosarcoma neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3417.

Characterization of a FOXG1:TLE1 transcriptional network in glioblastoma‐initiating cells

Glioblastoma (GBM) is the most common and deadly malignant brain cancer of glial cell origin, with a median patient survival of less than 20 months. Transcription factors FOXG1 and TLE1 promote GBM propagation by supporting maintenance of brain tumour‐initiating cells (BTICs) with stem‐like properties. Here, we characterize FOXG1 and TLE1 target genes in GBM patient‐derived BTICs using ChIP‐Seq and RNA‐Seq approaches. These studies identify 150 direct FOXG1 targets, several of which are also TLE1 targets, involved in cell proliferation, differentiation, survival, chemotaxis and angiogenesis. Negative regulators of NOTCH signalling, including CHAC1, are among the transcriptional repression targets of FOXG1:TLE1 complexes, suggesting a crosstalk between FOXG1:TLE1 and NOTCH‐mediated pathways in GBM. These results provide previously unavailable insight into the transcriptional programs underlying the tumour‐promoting functions of FOXG1:TLE1 in GBM.

Regulation of glioblastoma stem-like cell self-renewal by FOXG1 and TLE

Solomon Snyder Dept. of Neuroscience, Johns Hopkins School of Medicine, United States E-mail address: jdemelo@jhmi.edu (J. de Melo). The LIM homeodomain transcription factor Lhx2 is an essential factor for the development of the mammalian retina. Lhx2 knockout mice are embryonic lethal and display complete anophthalmia with arrest of ocular development occurring at the optic vesicle stage. We demonstrate that Lhx2 is expressed in mitotically active retinal progenitors during embryonic development. Expression of Lhx2 becomes restricted to Müller glia (MG) and a subset of amacrine cells in the post-natal retina within which Lhx2 expression remains throughout adulthood. Utilizing a conditional Lhx2 knockout mouse we have demonstrated that loss of Lhx2 at approximately E10 results in mitotic arrest and microphthalmia. Deletion of Lhx2 in Muller glial precursors, however, resulted in a failure in MG formation resulting in laminar disruption and rosette formation in the outer nuclear layer (ONL). In vivo electroporation experiments were performed to generate retinas in which Lhx2 was deleted in a mosaic fashion. Electroporation of plasmids expressing Cre recombinase into floxed Lhx2 mice replicated the MG phenotype seen in the conditional knockout mice. Electroporation of the pro-glial bHLH transcription factor Hes5 promoted formation of MG; however, co-electroporation of Hes5 with cre into floxed Lhx2 mice blocked the enhanced MG formation. Furthermore, co-electroporation of Lhx2 with Hes5 into wild-type retinas also blocked the formation of MG. While loss of Lhx2 function resulted in failed MG development, electroporation of Lhx2 alone did not promote the formation of MG but instead resulted in the generation of wide-field amacrine cells. Intriguingly, generation of wide field amacrine cells was enhanced by co-electroporating Lhx2 with the bHLH transcription factor Neurog2. Taken together these results suggest a model where Lhx2 maintains the retinal progenitor state and is instructive for the generation of wide-field amacrine cells. However, upon commitment to a MG fate Lhx2 is absolutely required for the differentiation of MG.

Abstract 5347: Inhibition of Notch3 signaling reduces tumorigenic properties of human rhabdomyosarcoma cells

Rhabdomyosarcoma is a soft tissue sarcoma of childhood arising from impaired developmental processes involving skeletal muscle progenitors. Notch signaling is strictly implicated in muscle tissue determination and has been reported to be abnormally activated in rhabdomyosarcoma. The expression of a dominant negative form of the Notch target gene HES1 or the inhibition of the γ-secretase has been shown to arrest rhabdomyosarcoma cell growth and trigger differentiation in vitro. Nevertheless, the mechanism underlying the deregulation of Notch signaling in rhabdomyosarcoma remains unclear. Here, we show that Notch3 and Notch1 are aberrantly activated in primary rhabdomyosarcomas and cell lines compared to skeletal muscle tissues or cultured myoblasts. In the present study we provide evidence that Notch3 targeted down-regulation restrains cell proliferation in both alveolar and embryonal subtypes of rhabdomyosarcoma cell lines. This phenomenon is paralleled by a transcriptional down-regulation of HES1 and an increase in both the cell cycle inhibitor p21cip1 and the under-phosphorylated pRb. Further, cells in which Notch3 has been silenced are capable to form differentiated multinucleated fibers showing de novo expression of late markers of myogenesis. In addition, down-regulation of Notch3 results in p38 and Akt hyper-phosphorylation and over-expression of PTEN. Finally, sustained silencing of Notch3 in rhabdomyosarcoma cells hampers their tumorigenic properties in vitro and in vivo. These results support a pathogenetic role for Notch3 in rhabdomyosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2011-5347