Factors Affecting EWS-FLI1 Activity in  Ewing's Sarcoma

David Herrero-Martin; Argyro Fourtouna; Stephan Niedan; Lucia T. Riedmann; Raphaela Schwentner; Dave N. T. Aryee

doi:10.1155/2011/352580

Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma

Herrero-Martin, David;Fourtouna, Argyro;Niedan, Stephan;Riedmann, Lucia T.;Schwentner, Raphaela;Aryee, Dave N. T. 2011-11-10 00:00:00 Hindawi Publishing Corporation Sarcoma Volume 2011, Article ID 352580, 11 pages doi:10.1155/2011/352580 Review Article David Herrero-Martin, Argyro Fourtouna, Stephan Niedan, Lucia T. Riedmann, Raphaela Schwentner, and Dave N. T. Aryee Children’s Cancer Research Institute, St Anna Kinderkrebsforschung, 1090 Vienna, Austria Correspondence should be addressed to David Herrero-Martin, david.herrero@ccri.at Received 15 July 2011; Revised 31 August 2011; Accepted 31 August 2011 Academic Editor: Alessandro Gronchi Copyright © 2011 David Herrero-Martin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ewing’s sarcoma family tumors (ESFT) are characterized by speciﬁc chromosomal translocations, which give rise to EWS-ETS chimeric proteins. These aberrant transcription factors are the main pathogenic drivers of ESFT. Elucidation of the factors inﬂuencing EWS-ETS expression and/or activity will guide the development of novel therapeutic agents against this fatal disease. 1. Introduction eﬀorts have been made to understand the function of this fusion protein. Knowledge about the detailed EWS-FLI1 ESFT comprise a group of undiﬀerentiated, highly malignant protein structure would be extremely helpful to analyse and small blue round-cell pediatric tumors. They are genetically predict its DNA-binding properties as a basis for a better characterized by EWS-ETS gene rearrangements aﬀecting understanding of the EWS-FLI1 transcriptional network and EWSR1 and genes of the ETS-family of transcription fac- for the development of inhibitory modalities with therapeu- tors, predominantly FLI1 characterizing 85% of cases [1]. tic promise. Numerous eﬀorts have been undertaken in the last three EWS-fusion proteins contain at least the N-terminal decades to explore the functional role of EWS-FLI1 in tumor 7exons of EWS comprising the EWS activation domain pathogenesis. EWS-FLI1 has been identiﬁed as the main (EAD). The EAD structure consists of multiple degenerate genetic factor of malignancy in ESFT [2, 3] and it is also hexapeptide repeats (consensus SYGQQS) with a conserved causal in the pathogenesis of ESFT from its cellular types tyrosine residue. However, systematic mutagenesis of the of origin [4–6]. It has been widely documented that this EAD revealed that the overall sequence composition and chimeric protein acts both as a transcriptional activator not the speciﬁc sequence of the degenerate hexapeptide and repressor of similarly sized sets of target genes [7, 8]. repeat confer EAD activity [72]. TheC-terminalportion While most mechanistic studies have concentrated on the of EWS-FLI1 consists of a COOH-terminal domain as identiﬁcation and description of downstream EWS-FLI1 well as an ets-type winged helix-lop-helix DNA-binding regulated genes, this paper focuses on currently known domain (DBD). Arvand et al. suggested that, in addition factors inﬂuencing EWS-FLI1 activity up- and downstream to the EAD and DBD domains, the COOH-terminal FLI1 of the fusion protein and consequently modulate its target domain contributes to promote cellular transformation [73]. gene expression. Mutation analysis of the EWS-DBD revealed that EWS-FLI1, apparently, not only induces DBD-dependent but also DBD- 2. Structure and Posttranslational independent oncogenic pathways, suggesting that EWS-FLI1 interacts with other gene regulatory factors or complexes Modiﬁcations Affecting the Transcriptional [74]. Activity of EWS-FLI1 Transcriptional regulation is tightly controlled by tran- Since, due to its tumor-speciﬁc expression, EWS-FLI1 pro- scription factor binding to regulatory regions within DNA as tein is considered an ideal therapeutic target [71], signiﬁcant well as recruitment of cofactors. Although ETS transcription 2 Sarcoma factors bind predominantly as monomers to a GGAA/T structure when isolated. A characteristic composition of core motif in promoter or enhancer regions of their target amino acids prevents these proteins from forming singular, genes, functional interaction between ETS proteins and other ﬁxed structures thereby enabling them for rapid complex factors is crucial to enhance or modulate DNA binding formation and dissociation with relatively high speciﬁcity [75]. Even though EWS-FLI1 possesses protein interaction and low aﬃnity [76]. As no direct enzymatic activity has been domains such as SH2 or PDZ, the identiﬁed intrinsically ascribed to EWS-FLI1, it is necessary to identify interaction disordered protein regions may facilitate protein-protein partners of the fusion protein in order to learn more about complexes as explained in the next chapter [76]. the functional pathways in which it is involved and how to However, transcriptional control also involves complex modulate them therapeutically. upstream signaling pathways that converge on the posttrans- EWS-FLI1 is generally perceived as a transcriptional ac- lational modiﬁcation of transcription factors and their in- tivator [79–81]. Consistent with its transcriptional activator teracting cofactors. Phosphorylation and glycosylation are function, EWS-FLI1 associates with several proteins of two examples of posttranslationally modifying mechanisms the basal transcription machinery. Among them are RNA aﬀecting EWS-FLI1 activity. The EWS portion of about 20% polymerase II [15] and its core subunit hsRBP7 [16–18], of EWS-FLI1 fusion proteins (those that retain EWS amino CREB-binding protein (CBP)/p300 [19], and RNA hel- acids 256 to 285) contains a conserved calmodulin-binding icase A (RHA) [22]. The interaction with (CBP)/p300 was motif within the IQ domain with a phosphorylated internal demonstrated to be involved in the regulation of several bona Protein Kinase C recognition site at Ser 266 [9]. Mutation of ﬁde EWS-FLI1 targets like p21 [82] or matrix metallopro- this residue was enough to signiﬁcantly reduce DNA binding teinase (MMP-1) [83]. RHA is a modulator of transcription of EWS-FLI1 in vitro [9, 10]. Furthermore, EWS and EWS- as it interacts with CBP/p300 and RNA polymerase II. FLI1 are phosphorylated at Thr 79 in the N-terminal domain Interruption of this interaction induces apoptosis in vivo in response to DNA damage or mitogens [11]. Glycosylation and in vitro, a potential novel therapeutic strategy [22, 53]. is the enzymatic process that attaches glycans to proteins, Interaction with the putative tumor suppressor BARD1, lipids, or other organic molecules [77]. EWS-FLI1 was found that associates with the breast cancer susceptibility gene to undergo O-linked beta-N-acetylglucosaminylation (O- BRCA1, links EWS-FLI1 with proteins involved in genome GlcNAcylation). This modiﬁcation seems to be reciprocally surveillance, DNA repair, and checkpoint control [23]. It is related to phosphorylation and to inﬂuence the transcrip- likely that target site selectivity of EWS-FLI1 is mediated tional activation propensities of the fusion protein [12]. In via interaction with other sequence speciﬁc transcription addition, N-linked glycosylation was described as essential factors. Such an interaction has been described for FOS- to sustain ESFT cell growth. Interestingly, inhibition of JUN dimers, which bind to AP1 sequences synergizing with N-linked glycosylation decreased the expression of EWS- EWS-FLI1 in the regulation of a subset of EWS-FLI1 target FLI1 correlating to growth arrest [13]. The highly decreased genes including uridine phosphorylase [24]. Recent in silico expression levels of EWS-FLI1 observed after treatment with analyses reveal a signiﬁcant enrichment of E2F binding sites HMG-CoA reductase inhibitors (i.e., lovastatin) or N-linked in EWS-FLI1 upregulated genes suggesting an important glycosylation inhibitors (i.e., tunicamycin) were found to role of the E2F family of transcription factors in EWS- be due to the instability of de novo-synthesized fusion FLI mediated transcriptional regulation [8]. Whether EWS- protein [13, 52]. Lovastatin triggered diﬀerentiation and FLI1 actually physically interacts with E2Fs to accomplish induced apoptosis without causing cell cycle arrest through upregulation of the aﬀected genes or merely binds alongside the loss of an RB-regulated G1 checkpoint [52]. Although E2F transcription factors remains to be elucidated. EWS-FLI1 contains four potential sites for this type of In addition to transcriptional activation, an at least equal posttranslational modiﬁcation, no evidence for direct N- number of genes are downregulated by EWS-FLI1 as are glycosylation of the fusion protein could be obtained. There- upregulated [25]. One explanation for this fact is that some fore, an indirect functional interaction involving other key- of the upregulated EWS-FLI1 targets are transcriptional player glycoproteins has been proposed [13]. Since block- repressors as exempliﬁed by NKX2.2, a directly EWS-FLI1- age of N-linked glycosylation also leads to inactivation of activated target which functions as a transcriptional repres- IGF-1R signaling by inhibiting translocation to the cell sor [84, 85]. Another target of EWS-FLI1, NR0B1, not only surface [14], and since IGF-1R activity is essential to EWS- acts as a transcriptional regulator downstream of EWS-FLI1 FLI1 expression (discussed in Section 4), inactivation of but also has recently been shown to interact physically with this pathway may at least partially explain why inhibi- EWS-FLI1 to inﬂuence gene expression thereby contributing tion of N-linked glycosylation leads to reduced expres- to Ewing’s sarcoma oncogenesis [25]. Due to interaction with sion of the fusion protein. However, further investiga- several RNA processing proteins including the small nuclear tions are required to test this hypothesis (summarized in ribonucleoprotein (snRNP) U1C [86], EWS-FLI1 activity Table 1). has not only been linked to RNA transcription but also to splicing [26, 87]. U1C plays a critical role in the initiation and regulation of pre-mRNA splicing as part of the U1 3. Direct EWS-FLI1 Protein Interactions small nuclear ribonucleoprotein and commits pre-mRNAs to Biochemical puriﬁcation and analysis identiﬁed EWS-FLI1 the splicing process [88]. Interestingly, forced U1C expres- sion was demonstrated to modulate dose—dependently the as an intrinsically disordered protein [72, 78]. Intrinsically disordered proteins are deﬁned by their lack of a stable transcriptional transactivation activity of EWS-FLI1 in vitro Sarcoma 3 Table 1: Factors inﬂuencing EWS-FLI1 activity and/or expression. Posttranslational modiﬁcations Phosphorylation DNA binding, response to DNA damage and mitogens [9–11] Glycosylation Transcriptional activation, cell growth, link with IGF-1 signaling [12–14] Direct protein-protein interactions RNA polymerase II Basal transcription machinery [15] hsRBP7 Basal transcription machinery [16–18] Basal transcription machinery, regulation of EWS-FLI1 targets like Creb-binding protein (CBP)/p300 [19–21] p21 or MMP-1 RNA helicase A (RHA) Modulator of transcription [22] Putative tumor suppressor; genome surveillance, DNA repair and BARD1 [23] checkpoint control Binding to AP1 sequences synergizing with EWS-FLI1, regulation of FOS-JUN dimers [24] uridine phosphorylase NR0B1 Transcriptional regulator downstream of EWS-FLI1 [25] small nuclear ribonucleoprotein (snRNP) U1C pre-mRNA splicing [26] EWS Functional consequences of this heterodimerization unknown [20] Factors indirectly aﬀecting EWS-FLI1 activity p53 and INK4A pathways Loss of each one stabilizes EWS-FLI1 [27–33] Hypoxia Apoptosis resistance via HIF, chemotherapy failure, angiogenesis [34–37] EWS-FLI1 mediated cellular transformation, proliferation and IGF-1/IGF-1R pathway [38–45] survival bFGF Triggers EWS-FLI1 expression in serum-depleted ESFT cells [46] BLCAP Ectopic overexpression decreases EWS-FLI1, apoptosis [47] miRNAs miR-145 EWS-FLI1 repressed miRNA, regulatory feedback loop [48, 49] miR-100, miR-125b, miR-22, miR-221/222, EWS-FLI1 repressed miRNAs, targets in IGF signalling pathway [50] miR-271 and miR29a let-7 family EWS-FLI1 repressed miRNA, let 7-a is a direct target of EWS-FLI1 [51] miRNA 17–92 cluster EWS-FLI1 induced miRNAs [51] and in vivo via interaction with the EWS amino terminal Consistent with this ﬁnding, loss of p53 greatly accelerates domain [86]. In addition, experimental evidence for a direct tumorigenesis in EWS-FLI1 transgenic mice [29]. However, interaction between EWS-FLI1 and EWS was reported by in ESFT, mutations in p53 or p16/p14ARF are found in Spahn et al. [20]. Since EWS interacts with a multitude of approximately 10% and 25% of cases, respectively. As in RNA processing factors [21], the functional consequences of most pediatric malignancies, the majority of ESFT express this heterodimerization on RNA splicing remains a subject wild-type p53 and p16/14ARF genes [30–32]. Functionally, forfurtherinvestigation (summarizedin Table 1). basal p53 expression is modulated by EWS-FLI1 through an indirect mechanism that involves suppression of the Notch signalling pathway [33]. 4. Factors Indirectly Affecting 4.2. Hypoxia. Hypoxia is a common condition in solid EWS-FLI1 Activity tumors. It drives cancer cells towards a coordinated set of 4.1. p53 and INK4A Pathways. The p53 and INK4A survival responses altering the transcriptional regulation of (p16/p14ARF) pathways are critical in promoting cell cycle many genes [91], stimulating cell migration, invasiveness arrest in response to mitogenic signals, and mutations in and motility [92], and driving a metabolic shift towards their key components facilitate tumor progression in most anaerobic glycolysis [93] or promotion of autophagy [94]. cancer types [89, 90]. In normal primary mouse ﬁbroblasts Due to its involvement in drug resistance [95], hypoxia (MEFs), EWS-FLI1 expression is unstable eliciting a p53- has been identiﬁed as a negative prognostic factor in many dependentgrowtharrestand apoptosisprogram.However, cancers [96] including sarcomas [97]. HIF-1, a basic HLH in p16 or p53 defective MEFs, these eﬀects are attenuated transcription factor, is a major player in the adaptive and this environment allows stable expression of the fusion response to hypoxic conditions, enhancing cell survival in protein [27, 28]. Thus, it appears that the loss of each of these this unfavourable environment [92–98]. In ESFT, hypoxia tumor suppressor genes stabilizes EWS-FLI1 expression. has been shown to contribute to apoptosis resistance via 4 Sarcoma HIF-1α [99], to chemotherapy resistance [34], and to the miRNA to a partially homologous region (seed region) establishment of an alternative circulatory system [35]. within the 3 untranslated region (UTR), coding sequences Interestingly, under hypoxic conditions, EWS-FLI1 pro- or 5 UTRs of messenger RNAs (mRNA), it can either block tein expression was demonstrated to increase transiently its target mRNA translation or lead to its degradation [102, in a HIF-1α-dependent manner [36]. HIF-1α-mediated 103]. Due to the imperfect base pairing of the miRNA to EWS-FLI1 accumulation involved protein regulation at the its seed region, a single miRNA can regulate several target FLI1 moiety, since the observed protein accumulation was mRNAsaspartofacomplexgeneregulatorynetwork [101, restricted to EWS-FLI1 and neither observed for full-length 104]. It is estimated that between 30% and 60% of the human EWS nor for an alternative EWS fusion to ERG. On the genome is regulated by miRNAs including genes involved transcriptional level, however, the upregulation of EWS-FLI1 in mechanisms of tumorigenesis, such as proliferation, protein did not simply result in a reinforcement of the EWS- inﬂammation, stress response, apoptosis, diﬀerentiation, and FLI1 transcriptional signature, but showed a more complex invasion [101, 102]. miRNAs can either act as oncogenes eﬀect with both synergistic and antagonistic consequences or tumor suppressors, some of them even in both ways on EWS-FLI1 regulated genes [36]. Another study has shown [101, 105, 106]. colocalisation of HIF-1α and necrotic areas in an ESFT tissue While the role of aberrantly expressed miRNAs is well array, suggesting a role for hypoxia in in vivo induction of established in adult cancers, only few studies exist for pe- HIF-1α [37]. Data thus implicates HIF as the main response diatric malignancies in general and sarcomas in particular factor for hypoxic stimulus in ESFT with marked eﬀects on [107–109]. One of the best described tumor suppressive proliferation and apoptosis. miRNAs is miR-145, which was found to be downregulated in several solid tumors, including lung, colorectal, breast, and prostate cancer [110, 111]. Similarly, in ESFT, miR- 4.3. IGF-1/IGF-1R and bFGF Pathways. The autocrine loops 145 was recently described as the top consistently EWS-FLI1 encompassing (IGF-1)/(IGF-1R) and (IGF-2)/(IGF-2R) play repressed miRNA. This ﬁnding was based on the investiga- a crucial role in the proliferation and survival of ESFT tion of ﬁve ESFT cell lines upon RNA interference-mediated cells via activation of AKT and ERK1/2 [38–40]. Notably, EWS-FLI1 knockdown and on diﬀerential gene expression in MEFs, expression of IGF-1R is required for EWS- patterns between primary ESFT and mesenchymal stem FLI1-mediated cellular transformation suggesting that the cells, the most related normal tissue. In fact, miR-145 and oncogenic activity of the fusion protein is dependent on EWS-FLI1 were demonstrated to build a regulatory feedback functional IGF-1R signaling [41]. There are several lines of loop, in which EWS-FLI1 suppresses miR-145 and miR- evidence that support a link between EWS-FLI1 and IGF- 145 modulates EWS-FLI1 expression [48, 49]. This type of 1/IGF1-R signalling [42, 43] also in one of the putative positive feedback regulation has the potential to serve as a progenitor cell of ESFT [44], and inhibition of this signaling compensating buﬀer for variations in EWS-FLI1 expression. pathway reduces tumor growth in vitro [45]and in vivo Reconstitution of miR-145 expression resulted in decreased [54], blocks angiogenesis [55], induces cell death [61], and EWS-FLI1 expression and consequently reduced cell growth increases chemosensitivity [100]. and soft agar colony formation [48]. Of note, miR-145 has A further growth factor positively interacting with EWS- recently been reported to target the 3 UTR of another ETS FLI1 activity is basic ﬁbroblast growth factor (bFGF). bFGF family gene, ERG, which replaces FLI in alternative EWS was demonstrated to trigger EWS-FLI1 expression in serum- fusions associated with about 10% of ESFT [112]. The DNA depleted ESFT cells. A neutralizing antibody against bFGF binding domain of ERG shares 98% homology with that of was able to disrupt this upregulation and inhibit expression FLI1 [113] and our own unpublished results suggest that of the fusion protein in a broad panel of ESFT cell lines there is signiﬁcant overlap between EWS-FLI1 target genes in [46]. No detectable eﬀect on EWS-FLI1 expression levels ESFT and ERG in prostate cancer cells. Although activity of was observed upon epidermal growth factor or platelet miR-145 on EWS-ERG in ESFT remains to be demonstrated, derived growth factor stimulation. However, the mechanism the ﬁnding of ERG modulation by this miRNA in prostate by which bFGF speciﬁcally controls EWS-FLI1 levels remains cancer cells may extend the concept of feedback regulation elusive between EWS-ETS fusion genes and miR-145 beyond EWS- Most recently, a further putative signalling molecule that FLI1. is expressed on the cell surface, the bladder cancer associated However, a recent global miRNA proﬁling study in the protein BLCAP, carrying a putative Ser-Pro-X-X motif and A673 ESFT cell line did not conﬁrm miR-145 among EWS- a proline-rich area, was reported to modulate EWS-FLI1 FLI1 suppressed miRNAs but described a group of EWS- expression [47]. The mechanism of this activity, which was FLI1 repressed miRNAs (miR-100, miR-125b, miR-22, miR- obtained upon artiﬁcial ectopic overexpression, remains to 221/222, miR-271, and miR29a) with predicted targets in be elucidated (summarized in Table 1). the IGF-1/IGF-1R signaling pathway [50], a key growth regulatory signaling pathway interacting with EWS-FLI1 expression/activity [41–43]. The lack of evidence for miR- 5. miRNAs Inﬂuencing EWS-FLI1 Activity 145 suppression in this study [50] as compared to the pre- MicroRNAs (miRNAs) are small (21–24 nucleotides), single- vious study [48] may be caused by the use of diﬀerent cell lines, diﬀerent screening platforms (Agilent-type micro- stranded, and noncoding RNAs that regulate gene expression in a variety of cellular processes [101]. By binding of the array versus Applied Biosystems quantitative stem loop Sarcoma 5 PCR), and/or the diﬀerent timing of miRNA screening after to hypoxia is critical for their survival and growth. Given EWS-FLI1 knockdown (10 days in [50] versus 4 days in the central role hypoxia plays in tumor progression and [48]). miR-145 is the ﬁrst miRNA shown to target FLI1 resistance to therapy, hypoxia might well be considered the and FLI1 fusion genes [48, 49, 110]. Given the length of best validated target that has yet to be exploited in oncology the FLI1 3 UTR (>2 kb), it is very likely that other miRNAs [137]. Some established drugs targeting hypoxia or the HIF- may have similar FLI1 and EWS-FLI1 modulatory activities 1 pathway (e.g., 2-methoxyestradiol, bortezomib) have been (summarized in Table 1). already tested in ESFT [63–65] and although bortezomib per se showed no clinical beneﬁt [138] and resistance appeared, 6. Therapeutic Potentials [139] the recent ﬁnding of hypoxia transiently enhancing EWS-FLI1 protein expression [36]may raisehopes for The existence of tumor-speciﬁc alterations in several can- a combined therapeutic window for ESFT patients with cers presents a unique opportunity for pharmacological new agents. Also, therapeutic strategies targeting the IGF- intervention to therapeutic beneﬁt. Although EWS-FLI1 1/IGF-1R loop may interfere with oncogenic functions of has only been identiﬁed in tumor cells and therefore EWS-FLI1. Antagonistic IGF-1R antibodies or small kinase provides a potential ideal therapeutic target, ESFT has so inhibitory molecules have been developed and are therefore far remained a targetable disease without a targeted drug currently tested in phase I/II clinical trials on ESFT patients [71, 114]. Suppression of EWS-FLI1 has been achieved by either alone [56–58] or in combination with the mTOR antisense technologies [115–121], small interfering RNA inhibitor temsirolimus [59] showing promising results. One (siRNA) [122–125], short hairpin RNA (shRNA) [42, 126– important fact is the status of the insulin receptor (IR) as 128], and small pharmacological compounds [53, 62]all ESFT patients with a low IGF-1R : IR ratio do not beneﬁt blocking the proliferation of ESFT cell lines and xenografted from anti-IGF-1R therapies [60]. A meta-analysis of small- tumors. Although some siRNA coupled to nanoparticles scale retrospective studies suggest that, although rare, ESFT have proved to be useful in preclinical models either alone harbouring p53 or p16/p14ARF mutations form a subset with [129–132] or combined with other therapeutic agents as particularly poor prognosis, highly aggressive behaviour, and rapamycin [133], the general lack of clinical translation of poor chemoresponse [140, 141]. Nutlin-3a, a small molecule some of these macromolecule-based strategies lies in the which antagonizes the interaction of MDM2 with p53, thus challenge of pharmacological delivery [134]. Being present stabilizing the tumor suppressor protein, is able to promote a only in tumor cells, directly targeting the activity of EWS- strong apoptotic arrest when applied to ESFT and showed a FLI1 by focusing on its protein-protein interactions, will synergistic eﬀect with other chemotherapeutic agents such as be a logical step towards identifying potential targets for etoposide, doxorubicin, vincristine, and actinomycin D in a developing eﬀective anti-ESFT therapies. Along this line, dose-dependent manner [66, 67]. As downstream targets of targeting binding partners essential for EWS-FLI1 oncogenic EWS-FLI1 have been reported to contribute to the oncogenic function holds promise in combating ESFT as has been activities of EWS-FLI1 [19, 25, 85, 126, 142–145], generating shown for RNA helicase A using the small molecule YK-4- compounds eﬀectively targeting these downstream eﬀectors 279 [53]. YK-4-279 blocked RNA helicase A binding to EWS- hold potential therapeutic beneﬁts as has been shown with FLI1, induced apoptosis in ESFT cell lines and also reduced ET-743 [68], Mithramycin [62], and ARA-C [69] although growth in ESFT xenografts. YK-4-279 can also target a it is necessary to be cautious as, for example, ARA-C has subpopulation of chemoresistant ESFT stem cells [135] and it shown minimal activity and hematologic toxicity in a phase has been recently described as an eﬀective antiinvasive agent II clinical trial [70]. Methods to evaluate the speciﬁcity, in ETV1 and ERG fusion positive prostate tumors although the mechanism of action of YK-4-279 in prostate cancer cells toxicity, metabolism, and excretion as well as adsorption and seems to be diﬀerent [136]. A further evaluation of this new distribution within tumor cells are warranted to advance role of YK-4-279 in ESFT would be needed. O-linked beta- these potential drugs into clinical trials (summarized in N-acetylglucosamine (O-GlcNAc), which modiﬁes nuclear Table 2). and cytoplasmic proteins on serine and threonine residues, was delineated to serine/threonine residues of the amino- 7. Conclusion terminal EWS transcriptional-activation domain of the EWS-FLI1 fusion protein by our laboratory. Inhibition of While attempts to understand the pathobiology of ESFT EWS-FLI1 O-GlcNAcylation interfered with transactivation have focused mainly on identifying EWS-FLI1 target genes of its target gene Id2 [12]. A better understanding of EWS- and downstream pathways, there are still many important FLI1 O-GlcNAcylation as it relates to gene transcription and unresolved questions regarding factors modulating EWS- the physiological mechanisms behind this process is likely to FLI1 activity. Manipulation of these factors may oﬀer lead to novel therapies for treating ESFT. Recently, our group therapeutic promise since it is diﬃcult to directly target a identiﬁed a positive feedback regulation between EWS-FLI1 transcription factor. This may be achieved by applying high and miR-145 as an important component of EWS-FLI1 throughput compound screening technologies as has been mediated tumorigenesis [48]. As such, targeting miR-145 or other miRNAs found to aﬀect EWS-FLI1 activity may performed to block EWS-FLI1 interaction with RNA helicase A[22, 53], and in EWS-FLI1 signature-based approaches as serve as a promising therapy strategy to improve the clinical outcome of ESFT patients. Also, the adaptation of tumors in the case of Mithramycin [62]. Such compounds may be 6 Sarcoma Table 2: Therapeutic agents targeting partners essential for EWS-FLI1. Name Characteristics Eﬀects Reference EWS-FLI1 expression, growth arrest, inactivation Mevalonate, tunicamycin Inhibitors of N-linked glycosylation [12–14] of IGF-1R signaling Triggering of diﬀerentiation, induction of Lovastatin HMG-CoA reductase inhibitor [14, 52] apoptosis, inactivation of IGF-1R signaling Blocking RNA helicase A binding to Induction of apoptosis in vitro and reduction of YK-4-279 [53] EWS-FLI1 growth in vivo Tumor growth reduction in vitro and in vivo, [45, 54– Anti-IGF-1R antibodies Blocking IGF-1/IGF-1R pathway angiogenesis blockage, cell death induction and 60] chemosensitivity increase Epigallocatechin gallate IGF-1R inhibitor, catechin derivative Blocks proliferation and induces cell death [61] Neutralizing antibody EWS-FLI1 downregulation through inhibition of Blocking bFGF pathway [46] against bFGF FGFR phosphorylation EWS-FLI1 inhibitor, decreases tumor growth in Mithramycin DNA binding transcriptional inhibitor [62] vitro and in vivo 2-methoxyestradiol, Induction of apoptosis, autophagy and cell cycle Inhibitors of hypoxia and/or HIF-1 pathway [63–65] bortezomib arrest in vitro Small molecule which antagonizes the Stabilization of p53, apoptotic arrest, synergistic Nutlin-3a [66, 67] interaction of MDM2 with p53 eﬀect with other chemotherapeutic agents Binds and alkylates DNA at the N2 position Induction of apoptosis, reduction of the activity Ecteinascidin 743 [68] of guanine of EWS-FLI1 targets EWS-FLI1 protein reduction, decrease of cell ARA-C (cytosine Antimetabolite, inhibitor of EWS-FLI1 viability, transformation and tumor growth in [69, 70] arabinoside) vivo Restored Let-7a expression resulted in ESFT Synthetic Let-7a Synthetic miRNA [51] growth inhibition in vivo more speciﬁc and highly eﬀective in neutralising EWS-FLI1 O-GlcNAcylation: O-linked beta-N-acetylglucosaminylation activity in ESFT cells with minimal toxicity. HMG-CoA: 3-Hydroxy-3-methylglutaryl-coenzyme The list of agents inﬂuencing EWS-FLI1 fusion protein activity and/or expression is consistently enriched. Some of A RB: Retinoblastoma them show crosstalk, as has been demonstrated between a group of EWS-FLI1 repressed miRNAs and targets of IGF- IGF-1: Insulin-like growth factor 1 1/IGF-1R pathway [50]. Since very little is known about IGF-1R: Insulin-like growth factor receptor 1 CBP: Creb-binding protein the inﬂuence of miRNAs on EWS-FLI1 activity, employing high-throughput screening assays to identify miRNAs with RHA: RNAhelicaseA speciﬁc eﬀects on EWS-FLI1 activity will provide additional MMP-1: Matrix metalloproteinase BARD1: BRCA1-associated RING domain targets for therapeutic development. Recently, the use of miRNA arrays to compare the miRNA expression proﬁle protein 1 snRNP: Small nuclear ribonucleoprotein of human mesenchymal stem cells (MSCs) and ESFT cell MEFs: Mouse embryonic ﬁbroblasts lines has shown induction of the oncogenic miRNA 17–92 cluster and repression of the tumor suppressor let-7 family. HIF-1: Hypoxia-inducible factor-1 bFGF: Basic ﬁbroblast growth factor Importantly, the feasibility of delivery of synthetic miRNA in vivo to achieve tumor growth inhibition was demonstrated IGF-2: Insulin-like growth factor 2 in this study [51]. For a better understanding of the interplay IGF-2R: Insulin-like growth factor receptor 2 miRNA: MicroRNA between the discussed factors, it should be crucial to also consider the individual clinical proﬁles of ESFT patients. UTR: 3 untranslated region mRNA: Messenger RNA shRNA: Short hairpin RNA Abbreviations O-GlcNAc: O-linked beta-N-acetylglucosamine ESFT: Ewing’s sarcoma family tumors ARA-C: Cytosine arabinoside EAD: EWS activation domain IR: Insulin receptor DBD: DNA-binding domain MSCs: Human mesenchymal stem cells BLCAP: Bladder cancer-associated protein. SH2: Src Homology 2 Sarcoma 7 Acknowledgments [15] L. Yang, H. A. Chansky, and D. D. Hickstein, “EWS·Fli- 1 fusion protein interacts with hyperphosphorylated RNA The authors would like to thank Dr. Heinrich Kovar for polymerase II and interferes with serine-arginine protein- constructive discussions and critical comments. All authors mediated RNA splicing,” The Journal of Biological Chemistry, vol. 275, no. 48, pp. 37612–37618, 2000. have contributed equally to the paper. [16] R. Petermann, B. M. Mossier, D. N. T. Aryee, V. Khazak, E. A. Golemis, and H. 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Publisher: Hindawi Publishing Corporation
Copyright: Copyright © 2011 David Herrero-Martin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN: 1357-714X
eISSN: 1369-1643
DOI: 10.1155/2011/352580
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Abstract

Hindawi Publishing Corporation Sarcoma Volume 2011, Article ID 352580, 11 pages doi:10.1155/2011/352580 Review Article David Herrero-Martin, Argyro Fourtouna, Stephan Niedan, Lucia T. Riedmann, Raphaela Schwentner, and Dave N. T. Aryee Children’s Cancer Research Institute, St Anna Kinderkrebsforschung, 1090 Vienna, Austria Correspondence should be addressed to David Herrero-Martin, david.herrero@ccri.at Received 15 July 2011; Revised 31 August 2011; Accepted 31 August 2011 Academic Editor: Alessandro Gronchi Copyright © 2011 David Herrero-Martin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ewing’s sarcoma family tumors (ESFT) are characterized by speciﬁc chromosomal translocations, which give rise to EWS-ETS chimeric proteins. These aberrant transcription factors are the main pathogenic drivers of ESFT. Elucidation of the factors inﬂuencing EWS-ETS expression and/or activity will guide the development of novel therapeutic agents against this fatal disease. 1. Introduction eﬀorts have been made to understand the function of this fusion protein. Knowledge about the detailed EWS-FLI1 ESFT comprise a group of undiﬀerentiated, highly malignant protein structure would be extremely helpful to analyse and small blue round-cell pediatric tumors. They are genetically predict its DNA-binding properties as a basis for a better characterized by EWS-ETS gene rearrangements aﬀecting understanding of the EWS-FLI1 transcriptional network and EWSR1 and genes of the ETS-family of transcription fac- for the development of inhibitory modalities with therapeu- tors, predominantly FLI1 characterizing 85% of cases [1]. tic promise. Numerous eﬀorts have been undertaken in the last three EWS-fusion proteins contain at least the N-terminal decades to explore the functional role of EWS-FLI1 in tumor 7exons of EWS comprising the EWS activation domain pathogenesis. EWS-FLI1 has been identiﬁed as the main (EAD). The EAD structure consists of multiple degenerate genetic factor of malignancy in ESFT [2, 3] and it is also hexapeptide repeats (consensus SYGQQS) with a conserved causal in the pathogenesis of ESFT from its cellular types tyrosine residue. However, systematic mutagenesis of the of origin [4–6]. It has been widely documented that this EAD revealed that the overall sequence composition and chimeric protein acts both as a transcriptional activator not the speciﬁc sequence of the degenerate hexapeptide and repressor of similarly sized sets of target genes [7, 8]. repeat confer EAD activity [72]. TheC-terminalportion While most mechanistic studies have concentrated on the of EWS-FLI1 consists of a COOH-terminal domain as identiﬁcation and description of downstream EWS-FLI1 well as an ets-type winged helix-lop-helix DNA-binding regulated genes, this paper focuses on currently known domain (DBD). Arvand et al. suggested that, in addition factors inﬂuencing EWS-FLI1 activity up- and downstream to the EAD and DBD domains, the COOH-terminal FLI1 of the fusion protein and consequently modulate its target domain contributes to promote cellular transformation [73]. gene expression. Mutation analysis of the EWS-DBD revealed that EWS-FLI1, apparently, not only induces DBD-dependent but also DBD- 2. Structure and Posttranslational independent oncogenic pathways, suggesting that EWS-FLI1 interacts with other gene regulatory factors or complexes Modiﬁcations Affecting the Transcriptional [74]. Activity of EWS-FLI1 Transcriptional regulation is tightly controlled by tran- Since, due to its tumor-speciﬁc expression, EWS-FLI1 pro- scription factor binding to regulatory regions within DNA as tein is considered an ideal therapeutic target [71], signiﬁcant well as recruitment of cofactors. Although ETS transcription 2 Sarcoma factors bind predominantly as monomers to a GGAA/T structure when isolated. A characteristic composition of core motif in promoter or enhancer regions of their target amino acids prevents these proteins from forming singular, genes, functional interaction between ETS proteins and other ﬁxed structures thereby enabling them for rapid complex factors is crucial to enhance or modulate DNA binding formation and dissociation with relatively high speciﬁcity [75]. Even though EWS-FLI1 possesses protein interaction and low aﬃnity [76]. As no direct enzymatic activity has been domains such as SH2 or PDZ, the identiﬁed intrinsically ascribed to EWS-FLI1, it is necessary to identify interaction disordered protein regions may facilitate protein-protein partners of the fusion protein in order to learn more about complexes as explained in the next chapter [76]. the functional pathways in which it is involved and how to However, transcriptional control also involves complex modulate them therapeutically. upstream signaling pathways that converge on the posttrans- EWS-FLI1 is generally perceived as a transcriptional ac- lational modiﬁcation of transcription factors and their in- tivator [79–81]. Consistent with its transcriptional activator teracting cofactors. Phosphorylation and glycosylation are function, EWS-FLI1 associates with several proteins of two examples of posttranslationally modifying mechanisms the basal transcription machinery. Among them are RNA aﬀecting EWS-FLI1 activity. The EWS portion of about 20% polymerase II [15] and its core subunit hsRBP7 [16–18], of EWS-FLI1 fusion proteins (those that retain EWS amino CREB-binding protein (CBP)/p300 [19], and RNA hel- acids 256 to 285) contains a conserved calmodulin-binding icase A (RHA) [22]. The interaction with (CBP)/p300 was motif within the IQ domain with a phosphorylated internal demonstrated to be involved in the regulation of several bona Protein Kinase C recognition site at Ser 266 [9]. Mutation of ﬁde EWS-FLI1 targets like p21 [82] or matrix metallopro- this residue was enough to signiﬁcantly reduce DNA binding teinase (MMP-1) [83]. RHA is a modulator of transcription of EWS-FLI1 in vitro [9, 10]. Furthermore, EWS and EWS- as it interacts with CBP/p300 and RNA polymerase II. FLI1 are phosphorylated at Thr 79 in the N-terminal domain Interruption of this interaction induces apoptosis in vivo in response to DNA damage or mitogens [11]. Glycosylation and in vitro, a potential novel therapeutic strategy [22, 53]. is the enzymatic process that attaches glycans to proteins, Interaction with the putative tumor suppressor BARD1, lipids, or other organic molecules [77]. EWS-FLI1 was found that associates with the breast cancer susceptibility gene to undergo O-linked beta-N-acetylglucosaminylation (O- BRCA1, links EWS-FLI1 with proteins involved in genome GlcNAcylation). This modiﬁcation seems to be reciprocally surveillance, DNA repair, and checkpoint control [23]. It is related to phosphorylation and to inﬂuence the transcrip- likely that target site selectivity of EWS-FLI1 is mediated tional activation propensities of the fusion protein [12]. In via interaction with other sequence speciﬁc transcription addition, N-linked glycosylation was described as essential factors. Such an interaction has been described for FOS- to sustain ESFT cell growth. Interestingly, inhibition of JUN dimers, which bind to AP1 sequences synergizing with N-linked glycosylation decreased the expression of EWS- EWS-FLI1 in the regulation of a subset of EWS-FLI1 target FLI1 correlating to growth arrest [13]. The highly decreased genes including uridine phosphorylase [24]. Recent in silico expression levels of EWS-FLI1 observed after treatment with analyses reveal a signiﬁcant enrichment of E2F binding sites HMG-CoA reductase inhibitors (i.e., lovastatin) or N-linked in EWS-FLI1 upregulated genes suggesting an important glycosylation inhibitors (i.e., tunicamycin) were found to role of the E2F family of transcription factors in EWS- be due to the instability of de novo-synthesized fusion FLI mediated transcriptional regulation [8]. Whether EWS- protein [13, 52]. Lovastatin triggered diﬀerentiation and FLI1 actually physically interacts with E2Fs to accomplish induced apoptosis without causing cell cycle arrest through upregulation of the aﬀected genes or merely binds alongside the loss of an RB-regulated G1 checkpoint [52]. Although E2F transcription factors remains to be elucidated. EWS-FLI1 contains four potential sites for this type of In addition to transcriptional activation, an at least equal posttranslational modiﬁcation, no evidence for direct N- number of genes are downregulated by EWS-FLI1 as are glycosylation of the fusion protein could be obtained. There- upregulated [25]. One explanation for this fact is that some fore, an indirect functional interaction involving other key- of the upregulated EWS-FLI1 targets are transcriptional player glycoproteins has been proposed [13]. Since block- repressors as exempliﬁed by NKX2.2, a directly EWS-FLI1- age of N-linked glycosylation also leads to inactivation of activated target which functions as a transcriptional repres- IGF-1R signaling by inhibiting translocation to the cell sor [84, 85]. Another target of EWS-FLI1, NR0B1, not only surface [14], and since IGF-1R activity is essential to EWS- acts as a transcriptional regulator downstream of EWS-FLI1 FLI1 expression (discussed in Section 4), inactivation of but also has recently been shown to interact physically with this pathway may at least partially explain why inhibi- EWS-FLI1 to inﬂuence gene expression thereby contributing tion of N-linked glycosylation leads to reduced expres- to Ewing’s sarcoma oncogenesis [25]. Due to interaction with sion of the fusion protein. However, further investiga- several RNA processing proteins including the small nuclear tions are required to test this hypothesis (summarized in ribonucleoprotein (snRNP) U1C [86], EWS-FLI1 activity Table 1). has not only been linked to RNA transcription but also to splicing [26, 87]. U1C plays a critical role in the initiation and regulation of pre-mRNA splicing as part of the U1 3. Direct EWS-FLI1 Protein Interactions small nuclear ribonucleoprotein and commits pre-mRNAs to Biochemical puriﬁcation and analysis identiﬁed EWS-FLI1 the splicing process [88]. Interestingly, forced U1C expres- sion was demonstrated to modulate dose—dependently the as an intrinsically disordered protein [72, 78]. Intrinsically disordered proteins are deﬁned by their lack of a stable transcriptional transactivation activity of EWS-FLI1 in vitro Sarcoma 3 Table 1: Factors inﬂuencing EWS-FLI1 activity and/or expression. Posttranslational modiﬁcations Phosphorylation DNA binding, response to DNA damage and mitogens [9–11] Glycosylation Transcriptional activation, cell growth, link with IGF-1 signaling [12–14] Direct protein-protein interactions RNA polymerase II Basal transcription machinery [15] hsRBP7 Basal transcription machinery [16–18] Basal transcription machinery, regulation of EWS-FLI1 targets like Creb-binding protein (CBP)/p300 [19–21] p21 or MMP-1 RNA helicase A (RHA) Modulator of transcription [22] Putative tumor suppressor; genome surveillance, DNA repair and BARD1 [23] checkpoint control Binding to AP1 sequences synergizing with EWS-FLI1, regulation of FOS-JUN dimers [24] uridine phosphorylase NR0B1 Transcriptional regulator downstream of EWS-FLI1 [25] small nuclear ribonucleoprotein (snRNP) U1C pre-mRNA splicing [26] EWS Functional consequences of this heterodimerization unknown [20] Factors indirectly aﬀecting EWS-FLI1 activity p53 and INK4A pathways Loss of each one stabilizes EWS-FLI1 [27–33] Hypoxia Apoptosis resistance via HIF, chemotherapy failure, angiogenesis [34–37] EWS-FLI1 mediated cellular transformation, proliferation and IGF-1/IGF-1R pathway [38–45] survival bFGF Triggers EWS-FLI1 expression in serum-depleted ESFT cells [46] BLCAP Ectopic overexpression decreases EWS-FLI1, apoptosis [47] miRNAs miR-145 EWS-FLI1 repressed miRNA, regulatory feedback loop [48, 49] miR-100, miR-125b, miR-22, miR-221/222, EWS-FLI1 repressed miRNAs, targets in IGF signalling pathway [50] miR-271 and miR29a let-7 family EWS-FLI1 repressed miRNA, let 7-a is a direct target of EWS-FLI1 [51] miRNA 17–92 cluster EWS-FLI1 induced miRNAs [51] and in vivo via interaction with the EWS amino terminal Consistent with this ﬁnding, loss of p53 greatly accelerates domain [86]. In addition, experimental evidence for a direct tumorigenesis in EWS-FLI1 transgenic mice [29]. However, interaction between EWS-FLI1 and EWS was reported by in ESFT, mutations in p53 or p16/p14ARF are found in Spahn et al. [20]. Since EWS interacts with a multitude of approximately 10% and 25% of cases, respectively. As in RNA processing factors [21], the functional consequences of most pediatric malignancies, the majority of ESFT express this heterodimerization on RNA splicing remains a subject wild-type p53 and p16/14ARF genes [30–32]. Functionally, forfurtherinvestigation (summarizedin Table 1). basal p53 expression is modulated by EWS-FLI1 through an indirect mechanism that involves suppression of the Notch signalling pathway [33]. 4. Factors Indirectly Affecting 4.2. Hypoxia. Hypoxia is a common condition in solid EWS-FLI1 Activity tumors. It drives cancer cells towards a coordinated set of 4.1. p53 and INK4A Pathways. The p53 and INK4A survival responses altering the transcriptional regulation of (p16/p14ARF) pathways are critical in promoting cell cycle many genes [91], stimulating cell migration, invasiveness arrest in response to mitogenic signals, and mutations in and motility [92], and driving a metabolic shift towards their key components facilitate tumor progression in most anaerobic glycolysis [93] or promotion of autophagy [94]. cancer types [89, 90]. In normal primary mouse ﬁbroblasts Due to its involvement in drug resistance [95], hypoxia (MEFs), EWS-FLI1 expression is unstable eliciting a p53- has been identiﬁed as a negative prognostic factor in many dependentgrowtharrestand apoptosisprogram.However, cancers [96] including sarcomas [97]. HIF-1, a basic HLH in p16 or p53 defective MEFs, these eﬀects are attenuated transcription factor, is a major player in the adaptive and this environment allows stable expression of the fusion response to hypoxic conditions, enhancing cell survival in protein [27, 28]. Thus, it appears that the loss of each of these this unfavourable environment [92–98]. In ESFT, hypoxia tumor suppressor genes stabilizes EWS-FLI1 expression. has been shown to contribute to apoptosis resistance via 4 Sarcoma HIF-1α [99], to chemotherapy resistance [34], and to the miRNA to a partially homologous region (seed region) establishment of an alternative circulatory system [35]. within the 3 untranslated region (UTR), coding sequences Interestingly, under hypoxic conditions, EWS-FLI1 pro- or 5 UTRs of messenger RNAs (mRNA), it can either block tein expression was demonstrated to increase transiently its target mRNA translation or lead to its degradation [102, in a HIF-1α-dependent manner [36]. HIF-1α-mediated 103]. Due to the imperfect base pairing of the miRNA to EWS-FLI1 accumulation involved protein regulation at the its seed region, a single miRNA can regulate several target FLI1 moiety, since the observed protein accumulation was mRNAsaspartofacomplexgeneregulatorynetwork [101, restricted to EWS-FLI1 and neither observed for full-length 104]. It is estimated that between 30% and 60% of the human EWS nor for an alternative EWS fusion to ERG. On the genome is regulated by miRNAs including genes involved transcriptional level, however, the upregulation of EWS-FLI1 in mechanisms of tumorigenesis, such as proliferation, protein did not simply result in a reinforcement of the EWS- inﬂammation, stress response, apoptosis, diﬀerentiation, and FLI1 transcriptional signature, but showed a more complex invasion [101, 102]. miRNAs can either act as oncogenes eﬀect with both synergistic and antagonistic consequences or tumor suppressors, some of them even in both ways on EWS-FLI1 regulated genes [36]. Another study has shown [101, 105, 106]. colocalisation of HIF-1α and necrotic areas in an ESFT tissue While the role of aberrantly expressed miRNAs is well array, suggesting a role for hypoxia in in vivo induction of established in adult cancers, only few studies exist for pe- HIF-1α [37]. Data thus implicates HIF as the main response diatric malignancies in general and sarcomas in particular factor for hypoxic stimulus in ESFT with marked eﬀects on [107–109]. One of the best described tumor suppressive proliferation and apoptosis. miRNAs is miR-145, which was found to be downregulated in several solid tumors, including lung, colorectal, breast, and prostate cancer [110, 111]. Similarly, in ESFT, miR- 4.3. IGF-1/IGF-1R and bFGF Pathways. The autocrine loops 145 was recently described as the top consistently EWS-FLI1 encompassing (IGF-1)/(IGF-1R) and (IGF-2)/(IGF-2R) play repressed miRNA. This ﬁnding was based on the investiga- a crucial role in the proliferation and survival of ESFT tion of ﬁve ESFT cell lines upon RNA interference-mediated cells via activation of AKT and ERK1/2 [38–40]. Notably, EWS-FLI1 knockdown and on diﬀerential gene expression in MEFs, expression of IGF-1R is required for EWS- patterns between primary ESFT and mesenchymal stem FLI1-mediated cellular transformation suggesting that the cells, the most related normal tissue. In fact, miR-145 and oncogenic activity of the fusion protein is dependent on EWS-FLI1 were demonstrated to build a regulatory feedback functional IGF-1R signaling [41]. There are several lines of loop, in which EWS-FLI1 suppresses miR-145 and miR- evidence that support a link between EWS-FLI1 and IGF- 145 modulates EWS-FLI1 expression [48, 49]. This type of 1/IGF1-R signalling [42, 43] also in one of the putative positive feedback regulation has the potential to serve as a progenitor cell of ESFT [44], and inhibition of this signaling compensating buﬀer for variations in EWS-FLI1 expression. pathway reduces tumor growth in vitro [45]and in vivo Reconstitution of miR-145 expression resulted in decreased [54], blocks angiogenesis [55], induces cell death [61], and EWS-FLI1 expression and consequently reduced cell growth increases chemosensitivity [100]. and soft agar colony formation [48]. Of note, miR-145 has A further growth factor positively interacting with EWS- recently been reported to target the 3 UTR of another ETS FLI1 activity is basic ﬁbroblast growth factor (bFGF). bFGF family gene, ERG, which replaces FLI in alternative EWS was demonstrated to trigger EWS-FLI1 expression in serum- fusions associated with about 10% of ESFT [112]. The DNA depleted ESFT cells. A neutralizing antibody against bFGF binding domain of ERG shares 98% homology with that of was able to disrupt this upregulation and inhibit expression FLI1 [113] and our own unpublished results suggest that of the fusion protein in a broad panel of ESFT cell lines there is signiﬁcant overlap between EWS-FLI1 target genes in [46]. No detectable eﬀect on EWS-FLI1 expression levels ESFT and ERG in prostate cancer cells. Although activity of was observed upon epidermal growth factor or platelet miR-145 on EWS-ERG in ESFT remains to be demonstrated, derived growth factor stimulation. However, the mechanism the ﬁnding of ERG modulation by this miRNA in prostate by which bFGF speciﬁcally controls EWS-FLI1 levels remains cancer cells may extend the concept of feedback regulation elusive between EWS-ETS fusion genes and miR-145 beyond EWS- Most recently, a further putative signalling molecule that FLI1. is expressed on the cell surface, the bladder cancer associated However, a recent global miRNA proﬁling study in the protein BLCAP, carrying a putative Ser-Pro-X-X motif and A673 ESFT cell line did not conﬁrm miR-145 among EWS- a proline-rich area, was reported to modulate EWS-FLI1 FLI1 suppressed miRNAs but described a group of EWS- expression [47]. The mechanism of this activity, which was FLI1 repressed miRNAs (miR-100, miR-125b, miR-22, miR- obtained upon artiﬁcial ectopic overexpression, remains to 221/222, miR-271, and miR29a) with predicted targets in be elucidated (summarized in Table 1). the IGF-1/IGF-1R signaling pathway [50], a key growth regulatory signaling pathway interacting with EWS-FLI1 expression/activity [41–43]. The lack of evidence for miR- 5. miRNAs Inﬂuencing EWS-FLI1 Activity 145 suppression in this study [50] as compared to the pre- MicroRNAs (miRNAs) are small (21–24 nucleotides), single- vious study [48] may be caused by the use of diﬀerent cell lines, diﬀerent screening platforms (Agilent-type micro- stranded, and noncoding RNAs that regulate gene expression in a variety of cellular processes [101]. By binding of the array versus Applied Biosystems quantitative stem loop Sarcoma 5 PCR), and/or the diﬀerent timing of miRNA screening after to hypoxia is critical for their survival and growth. Given EWS-FLI1 knockdown (10 days in [50] versus 4 days in the central role hypoxia plays in tumor progression and [48]). miR-145 is the ﬁrst miRNA shown to target FLI1 resistance to therapy, hypoxia might well be considered the and FLI1 fusion genes [48, 49, 110]. Given the length of best validated target that has yet to be exploited in oncology the FLI1 3 UTR (>2 kb), it is very likely that other miRNAs [137]. Some established drugs targeting hypoxia or the HIF- may have similar FLI1 and EWS-FLI1 modulatory activities 1 pathway (e.g., 2-methoxyestradiol, bortezomib) have been (summarized in Table 1). already tested in ESFT [63–65] and although bortezomib per se showed no clinical beneﬁt [138] and resistance appeared, 6. Therapeutic Potentials [139] the recent ﬁnding of hypoxia transiently enhancing EWS-FLI1 protein expression [36]may raisehopes for The existence of tumor-speciﬁc alterations in several can- a combined therapeutic window for ESFT patients with cers presents a unique opportunity for pharmacological new agents. Also, therapeutic strategies targeting the IGF- intervention to therapeutic beneﬁt. Although EWS-FLI1 1/IGF-1R loop may interfere with oncogenic functions of has only been identiﬁed in tumor cells and therefore EWS-FLI1. Antagonistic IGF-1R antibodies or small kinase provides a potential ideal therapeutic target, ESFT has so inhibitory molecules have been developed and are therefore far remained a targetable disease without a targeted drug currently tested in phase I/II clinical trials on ESFT patients [71, 114]. Suppression of EWS-FLI1 has been achieved by either alone [56–58] or in combination with the mTOR antisense technologies [115–121], small interfering RNA inhibitor temsirolimus [59] showing promising results. One (siRNA) [122–125], short hairpin RNA (shRNA) [42, 126– important fact is the status of the insulin receptor (IR) as 128], and small pharmacological compounds [53, 62]all ESFT patients with a low IGF-1R : IR ratio do not beneﬁt blocking the proliferation of ESFT cell lines and xenografted from anti-IGF-1R therapies [60]. A meta-analysis of small- tumors. Although some siRNA coupled to nanoparticles scale retrospective studies suggest that, although rare, ESFT have proved to be useful in preclinical models either alone harbouring p53 or p16/p14ARF mutations form a subset with [129–132] or combined with other therapeutic agents as particularly poor prognosis, highly aggressive behaviour, and rapamycin [133], the general lack of clinical translation of poor chemoresponse [140, 141]. Nutlin-3a, a small molecule some of these macromolecule-based strategies lies in the which antagonizes the interaction of MDM2 with p53, thus challenge of pharmacological delivery [134]. Being present stabilizing the tumor suppressor protein, is able to promote a only in tumor cells, directly targeting the activity of EWS- strong apoptotic arrest when applied to ESFT and showed a FLI1 by focusing on its protein-protein interactions, will synergistic eﬀect with other chemotherapeutic agents such as be a logical step towards identifying potential targets for etoposide, doxorubicin, vincristine, and actinomycin D in a developing eﬀective anti-ESFT therapies. Along this line, dose-dependent manner [66, 67]. As downstream targets of targeting binding partners essential for EWS-FLI1 oncogenic EWS-FLI1 have been reported to contribute to the oncogenic function holds promise in combating ESFT as has been activities of EWS-FLI1 [19, 25, 85, 126, 142–145], generating shown for RNA helicase A using the small molecule YK-4- compounds eﬀectively targeting these downstream eﬀectors 279 [53]. YK-4-279 blocked RNA helicase A binding to EWS- hold potential therapeutic beneﬁts as has been shown with FLI1, induced apoptosis in ESFT cell lines and also reduced ET-743 [68], Mithramycin [62], and ARA-C [69] although growth in ESFT xenografts. YK-4-279 can also target a it is necessary to be cautious as, for example, ARA-C has subpopulation of chemoresistant ESFT stem cells [135] and it shown minimal activity and hematologic toxicity in a phase has been recently described as an eﬀective antiinvasive agent II clinical trial [70]. Methods to evaluate the speciﬁcity, in ETV1 and ERG fusion positive prostate tumors although the mechanism of action of YK-4-279 in prostate cancer cells toxicity, metabolism, and excretion as well as adsorption and seems to be diﬀerent [136]. A further evaluation of this new distribution within tumor cells are warranted to advance role of YK-4-279 in ESFT would be needed. O-linked beta- these potential drugs into clinical trials (summarized in N-acetylglucosamine (O-GlcNAc), which modiﬁes nuclear Table 2). and cytoplasmic proteins on serine and threonine residues, was delineated to serine/threonine residues of the amino- 7. Conclusion terminal EWS transcriptional-activation domain of the EWS-FLI1 fusion protein by our laboratory. Inhibition of While attempts to understand the pathobiology of ESFT EWS-FLI1 O-GlcNAcylation interfered with transactivation have focused mainly on identifying EWS-FLI1 target genes of its target gene Id2 [12]. A better understanding of EWS- and downstream pathways, there are still many important FLI1 O-GlcNAcylation as it relates to gene transcription and unresolved questions regarding factors modulating EWS- the physiological mechanisms behind this process is likely to FLI1 activity. Manipulation of these factors may oﬀer lead to novel therapies for treating ESFT. Recently, our group therapeutic promise since it is diﬃcult to directly target a identiﬁed a positive feedback regulation between EWS-FLI1 transcription factor. This may be achieved by applying high and miR-145 as an important component of EWS-FLI1 throughput compound screening technologies as has been mediated tumorigenesis [48]. As such, targeting miR-145 or other miRNAs found to aﬀect EWS-FLI1 activity may performed to block EWS-FLI1 interaction with RNA helicase A[22, 53], and in EWS-FLI1 signature-based approaches as serve as a promising therapy strategy to improve the clinical outcome of ESFT patients. Also, the adaptation of tumors in the case of Mithramycin [62]. Such compounds may be 6 Sarcoma Table 2: Therapeutic agents targeting partners essential for EWS-FLI1. Name Characteristics Eﬀects Reference EWS-FLI1 expression, growth arrest, inactivation Mevalonate, tunicamycin Inhibitors of N-linked glycosylation [12–14] of IGF-1R signaling Triggering of diﬀerentiation, induction of Lovastatin HMG-CoA reductase inhibitor [14, 52] apoptosis, inactivation of IGF-1R signaling Blocking RNA helicase A binding to Induction of apoptosis in vitro and reduction of YK-4-279 [53] EWS-FLI1 growth in vivo Tumor growth reduction in vitro and in vivo, [45, 54– Anti-IGF-1R antibodies Blocking IGF-1/IGF-1R pathway angiogenesis blockage, cell death induction and 60] chemosensitivity increase Epigallocatechin gallate IGF-1R inhibitor, catechin derivative Blocks proliferation and induces cell death [61] Neutralizing antibody EWS-FLI1 downregulation through inhibition of Blocking bFGF pathway [46] against bFGF FGFR phosphorylation EWS-FLI1 inhibitor, decreases tumor growth in Mithramycin DNA binding transcriptional inhibitor [62] vitro and in vivo 2-methoxyestradiol, Induction of apoptosis, autophagy and cell cycle Inhibitors of hypoxia and/or HIF-1 pathway [63–65] bortezomib arrest in vitro Small molecule which antagonizes the Stabilization of p53, apoptotic arrest, synergistic Nutlin-3a [66, 67] interaction of MDM2 with p53 eﬀect with other chemotherapeutic agents Binds and alkylates DNA at the N2 position Induction of apoptosis, reduction of the activity Ecteinascidin 743 [68] of guanine of EWS-FLI1 targets EWS-FLI1 protein reduction, decrease of cell ARA-C (cytosine Antimetabolite, inhibitor of EWS-FLI1 viability, transformation and tumor growth in [69, 70] arabinoside) vivo Restored Let-7a expression resulted in ESFT Synthetic Let-7a Synthetic miRNA [51] growth inhibition in vivo more speciﬁc and highly eﬀective in neutralising EWS-FLI1 O-GlcNAcylation: O-linked beta-N-acetylglucosaminylation activity in ESFT cells with minimal toxicity. HMG-CoA: 3-Hydroxy-3-methylglutaryl-coenzyme The list of agents inﬂuencing EWS-FLI1 fusion protein activity and/or expression is consistently enriched. Some of A RB: Retinoblastoma them show crosstalk, as has been demonstrated between a group of EWS-FLI1 repressed miRNAs and targets of IGF- IGF-1: Insulin-like growth factor 1 1/IGF-1R pathway [50]. Since very little is known about IGF-1R: Insulin-like growth factor receptor 1 CBP: Creb-binding protein the inﬂuence of miRNAs on EWS-FLI1 activity, employing high-throughput screening assays to identify miRNAs with RHA: RNAhelicaseA speciﬁc eﬀects on EWS-FLI1 activity will provide additional MMP-1: Matrix metalloproteinase BARD1: BRCA1-associated RING domain targets for therapeutic development. Recently, the use of miRNA arrays to compare the miRNA expression proﬁle protein 1 snRNP: Small nuclear ribonucleoprotein of human mesenchymal stem cells (MSCs) and ESFT cell MEFs: Mouse embryonic ﬁbroblasts lines has shown induction of the oncogenic miRNA 17–92 cluster and repression of the tumor suppressor let-7 family. HIF-1: Hypoxia-inducible factor-1 bFGF: Basic ﬁbroblast growth factor Importantly, the feasibility of delivery of synthetic miRNA in vivo to achieve tumor growth inhibition was demonstrated IGF-2: Insulin-like growth factor 2 in this study [51]. For a better understanding of the interplay IGF-2R: Insulin-like growth factor receptor 2 miRNA: MicroRNA between the discussed factors, it should be crucial to also consider the individual clinical proﬁles of ESFT patients. UTR: 3 untranslated region mRNA: Messenger RNA shRNA: Short hairpin RNA Abbreviations O-GlcNAc: O-linked beta-N-acetylglucosamine ESFT: Ewing’s sarcoma family tumors ARA-C: Cytosine arabinoside EAD: EWS activation domain IR: Insulin receptor DBD: DNA-binding domain MSCs: Human mesenchymal stem cells BLCAP: Bladder cancer-associated protein. SH2: Src Homology 2 Sarcoma 7 Acknowledgments [15] L. Yang, H. A. Chansky, and D. D. Hickstein, “EWS·Fli- 1 fusion protein interacts with hyperphosphorylated RNA The authors would like to thank Dr. Heinrich Kovar for polymerase II and interferes with serine-arginine protein- constructive discussions and critical comments. All authors mediated RNA splicing,” The Journal of Biological Chemistry, vol. 275, no. 48, pp. 37612–37618, 2000. have contributed equally to the paper. [16] R. Petermann, B. M. Mossier, D. N. T. Aryee, V. Khazak, E. A. Golemis, and H. 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Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma

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Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma

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