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Clinical Cancer Research 13, 2429-2440, April 15, 2007. doi: 10.1158/1078-0432.CCR-06-1762
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Cholecystokinin Down-Regulation by RNA Interference Impairs Ewing Tumor Growth

Jaime Carrillo1, Eva García-Aragoncillo1, Daniel Azorín2, Noelia Agra1, Ana Sastre3, Imelda González-Mediero2, Purificación García-Miguel3, Ángel Pestaña1, Soledad Gallego4, Dolores Segura5 and Javier Alonso1

Authors' Affiliations: 1 Laboratorio de Patología Molecular de Tumores Sólidos Infantiles, Departamento de Biología Molecular y Celular del Cáncer, Instituto de Investigaciones Biomédicas "A. Sols" (CSIC-UAM); 2 Departamento Anatomía Patológica, Hospital Niño Jesús; 3 Unidad Oncohematología Pediátrica, Hospital Infantil La Paz, Madrid, Spain; 4 Unidad Hemato-Oncología Pediátrica, Hospital Vall d'Hebron, Barcelona, Spain; and 5 Departamento Anatomía Patológica, Hospital Virgen del Rocío, Sevilla, Spain

Requests for reprints: Javier Alonso, Laboratorio de Patología Molecular de Tumores Sólidos Infantiles, Departamento de Biología Molecular y Celular del Cáncer, Instituto de Investigaciones Biomédicas "A. Sols," CSIC-UAM, C/ Arturo Duperier 4, 28029 Madrid, Spain. Phone: 34-91-585-4418; Fax: 34-91-585-4401; E-mail: jalonso{at}iib.uam.es.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Tumors of the Ewing family are characterized by chromosomal translocations that yield chimeric transcription factors, such as EWS/FLI1, which regulate the expression of specific genes that contribute to the malignant phenotype. In the present study, we show that cholecystokinin (CCK) is a new target of the EWS/FLI1 oncoprotein and assess its functional role in Ewing tumor pathogenesis.

Experimental Design: Relevant EWS/FLI1 targets were identified using a combination of cell systems with inducible EWS/FLI1 expression, Ewing tumors and cell lines, microarrays, and RNA interference with doxycycline-inducible small hairpin RNA (shRNA) vectors. A doxycycline-inducible CCK-shRNA vector was stably transfected in A673 and SK-PN-DW Ewing cell lines to assess the role of CCK in cell proliferation and tumor growth.

Results: Microarray analysis revealed that CCK was up-regulated by EWS/FLI1 in HeLa cells. CCK was overexpressed in Ewing tumors as compared with other pediatric malignancies such as rhabdomyosarcoma and neuroblastoma, with levels close to those detected in normal tissues expressing the highest levels of CCK. Furthermore, EWS/FLI1 knockdown in A673 and SK-PN-DW Ewing cells using two different doxycycline-inducible EWS/FLI1-specific shRNA vectors down-regulated CCK mRNA expression and diminished the levels of secreted CCK, showing that CCK is a EWS/FLI1 specific target gene in Ewing cells. A doxycycline-inducible CCK-specific shRNA vector successfully down-regulated CCK expression, reduced the levels of secreted CCK in Ewing cell lines, and inhibited cell growth and proliferation in vitro and in vivo. Finally, we show that Ewing cell lines and tumors express CCK receptors and that the growth inhibition produced by CCK silencing can be rescued by culturing the cells with medium containing CCK.

Conclusions: Our data support the hypothesis that CCK acts as an autocrine growth factor stimulating the proliferation of Ewing cells and suggest that therapies targeting CCK could be promising in the treatment of Ewing tumors.

Ewing family of tumors is a group of highly malignant tumors arising mainly in bone that most often affects children and young adults in the first two decades of life. Despite the use of multimodal therapy (chemotherapy, radiation therapy, and surgery), the long-term disease-free survival rate of Ewing tumor patients is still disappointingly low, particularly in the high-risk groups with metastasis at diagnosis or unfavorable localization (reviewed in ref. 1). The lack of new effective drugs in the treatment of Ewing tumors, together with the side effects that high-dose regimens have on young patients who have a long life expectancy, supports the need for innovative therapeutic strategies that should mainly include targeted therapies against molecules critical for the pathogenesis and progression of these tumors.

The molecular hallmark of Ewing family of tumors is the presence of balanced chromosomal translocations leading to the formation of chimeric transcription factors. These aberrant proteins are formed by the NH2-terminal region of the RNA-binding protein EWS (or a related gene named TLS/FUS) and the COOH-terminal region of one of five members of the ETS family of transcription factors: FLI1, ERG, FEV, E1AF, and ETV1. The EWS/FLI1 combination is most frequent and is present in 85% of all cases of Ewing tumors (reviewed in refs. 2–4). Since 1992, when these translocations were discovered and characterized (5), a large body of evidence has shown the oncogenic potential of EWS/ETS proteins (6, 7). The most conclusive evidence for the essential tumorigenic role of EWS/FLI1 in Ewing cells comes from knockdown experiments. These studies have clearly shown that down-regulation of EWS/FLI1 using a variety of techniques efficiently inhibits the growth of Ewing tumor cells both in vitro and in vivo (8–11).

EWS/ETS fusion proteins retain the DNA binding motif characteristic of the ETS transcription factors, and thus the capability to bind to specific DNA sequences. Several studies have shown that preservation of the transcription factor activity is necessary for full tumorigenesis (6, 7, 12, 13), indicating that the oncogenic properties attributable to EWS/ETS are a consequence of the activation and repression of specific genes important for tumorigenesis. In recent years, several targets of the EWS/ETS proteins have been identified using both heterologous systems (non-Ewing related) and Ewing tumor cells. However, except for a small group of genes for which functional data are available (10, 14–17; reviewed in ref. 3), the contribution to Ewing pathogenesis remains undefined for the majority of the EWS/FLI1 target genes thus far identified. If a determined EWS/FLI1 target gene plays an important role in Ewing pathogenesis (e.g., promotion of cell proliferation or cell survival), it is conceivable that therapies targeting these genes should be effective in the treatment of these tumors.

We have used a combination of EWS/FLI1-inducible cell systems, Ewing tumors and cell lines, microarray, and RNA interference technologies to identify one relevant target of EWS/FLI1 and determine its functional role in the pathogenesis of Ewing tumors. Through this approach, we show that cholecystokinin (CCK), a neuroendocrine peptide involved in a wide range of biological functions including regulation of cell growth (18), is a relevant target of EWS/FLI1 in Ewing cells. Importantly, we show that silencing of CCK with RNA interference impairs cell proliferation and tumor growth in vivo. Our data support the idea that CCK acts as an autocrine growth factor stimulating the proliferation of Ewing cells and suggest that therapies targeting CCK could be helpful in the treatment of Ewing tumors.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and tumors. The Ewing tumor cell lines A673 (CRL-1598), SK-ES-1 (HTB-86), RD-ES (HTB-166), SK-N-MC (HTB-10), and SK-PN-DW (CRL-2139) were purchased from the American Type Culture Collection (Manassas, VA). Ewing tumor cell lines A4573 and TTC-466 were generous gifts from Dr. S. Navarro (University of Valencia, Valencia, Spain) and Dr. T.J. Triche (Children's Hospital Los Angeles, Los Angeles, CA), respectively. Tumors used in this study were provided by the Departments of Pathology and Oncology Units of several Spanish Children's Hospitals. This study was approved by the institutional review boards and informed consent was obtained from each patient's guardian.

Establishment of cell lines expressing inducible EWS/FLI1 and FLI1 proteins. Cloning of EWS/FLI1 and FLI1 cDNAs in expression vectors and establishment of cell lines are described in detail elsewhere (19). Briefly, we used two different cellular systems to achieve inducible and regulated expression of EWS/FLI1 and native FLI1. In the first one, the HeLa TeT-On cell line (BD Clontech, Palo Alto, CA), which constitutively expresses the reverse tetracycline-responsive transcriptional activator, was cotransfected with empty vector (HeLa pTET) or with pTET-EWS/FLI1 (HeLa pTET-E/F) or pTET-FLI1 (HeLa pTET-FLI1) vector together with the pTK-Hyg vector (BD Clontech), and then selected with the antibiotic hygromycin. In the second one, the 293 EcR cell line (Invitrogen, Paisley, United Kingdom), which constitutively expresses the ecdysone receptor (EcR), was transfected with pIND (empty vector, 293 EcR-pIND), pIND-EWS/FLI1 (293 EcR-E/F), or pIND-FLI1 (293 EcR-FLI1) vector, and selected with G418 to obtain stable transfectants. Induction of the EWS/FLI1 or FLI1 proteins was started by the addition of 1 µg/mL doxycycline (a tetracycline analogue) for the HeLa TeT-On system or 2.5 µmol/L ponasterone A (an ecdysone analogue) for the 293 EcR cell model. Expression of the EWS/FLI1 and FLI1 proteins was analyzed by Western blot with a rabbit polyclonal antibody raised against the COOH-terminal region of FLI1, present in both proteins (clone C-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

CodeLink microarrays for analysis of gene expression profiles. CodeLink Human Uniset I (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ) and CodeLink Human 20K Expression Bioarrays containing ~10,000 and 20,000 gene probes, respectively, which were derived from well-annotated mRNA sequences, were used to analyze gene expression profiles. Except where indicated, all reagents for labeling and hybridization were provided in the CodeLink expression assay reagent kit. RNA was isolated with TRI reagent (Sigma, St. Louis, MO) and its integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). Double-stranded cDNA and biotin-labeled cRNA were generated following the manufacturer's instructions, except that biotin-16-UTP (Roche Applied Science, Penzberg, Germany) was used instead of biotin-11-UTP. Biotin-labeled cRNA was purified on an RNeasy column (Qiagen, Valencia, CA), quantified by UV spectrophotometry, and analyzed for integrity using an Agilent 2100 Bioanalyzer. Then, 10 µg of cRNA were fragmented by heating at 94°C for 20 min in fragmentation buffer, subsequently diluted in hybridization buffer, and hybridized to CodeLink Bioarrays for 20 h at 37°C in an Innova 40 shaking incubator (New Brunswick, Edison, NJ) at 300 rpm. After hybridization, microarrays were washed in 0.75x TNT buffer [1x TNT: 0.15 mol/L NaCl, 0.05% Tween 20, 1 mol/L Tris-HCl (pH 7.6)] for 1 h at 46°C, incubated with Cy5-streptavidin for 30 min at room temperature, washed in 1x TNT four times for 5 min each, followed by two rinses in 0.1x SSC, 0.05% Tween 20, and then dried by centrifugation. Slides were scanned with an Axon GenePix Scanner (Arlington, TX) and analyzed using CodeLink Expression Analysis software (GE Healthcare).

To identify genes regulated by EWS/FLI1, we did two independent experiments. RNA from both HeLa pTET and HeLa pTET-E/F cells was isolated from unstimulated cells (basal gene expression profile at 0 h) and after 72 h of doxycycline stimulation, and subsequently labeled and hybridized to CodeLink Human Uniset I microarrays. After median normalization of microarray data, we calculated for each gene the ratio (HeLa pTET-E/F at 72 h/HeLa pTET-E/F at 0 h) versus (HeLa pTET at 72 h / HeLa pTET at 0 h). This ratio takes into account the unspecific effects that doxycycline and cell culture per se could have on gene expression. Genes in which this ratio was >2 in both experiments were considered to be up-regulated by EWS/FLI1 in HeLa cells.

To identify genes overexpressed in Ewing tumors in relation to neuroblastoma and rhabdomyosarcoma, the RNA isolated from tumors was labeled and hybridized to CodeLink Human 20K microarrays. After median normalization of microarray data, we calculated the median expression values from each gene in the Ewing tumor group and in the neuroblastoma and rhabdomyosarcoma group and calculated the ratio between the two values. We also calculated the P value using Student's t test with Bonferroni correction (which is a very stringent method) to establish the statistical differences between the two groups. We considered that a gene was overexpressed in Ewing tumors in relation to neuroblastoma and rhabdomyosarcoma when the ratio between groups was >5 and a statistical significance of P < 0.005 was reached using Student's t test.

Establishment of Ewing cell lines expressing doxycycline-inducible small hairpin RNA. We used the BLOCK-iT lentiviral expression system (Invitrogen) to establish Ewing cell lines harboring doxycycline-inducible small hairpin RNAs (shRNA). In this system, shRNA synthesis is driven by a H1 promoter, in which two canonical tetracycline repressor binding sites have been inserted downstream of the H1 promoter to achieve regulated and inducible expression of the shRNA on doxycycline stimulation. Lentiviruses were generated in 293-FT packaging cells transfected with lentiviral vectors and ViraPower packaging mix (Invitrogen) according to the manufacturer's instructions. Forty-eight hours posttransfection, the viral supernatant was used to infect the cells in the presence of polybrene (6 µg/mL).

We first engineered the A673 and SK-PN-DW Ewing cell lines to constitutively express high levels of the tetracycline repressor (TR). Briefly, A673 and SK-PN-DW cells were infected with high titers of lentiviruses containing the pLenti6/TR expression plasmid. After selection with blasticidin (3 µg/mL), constitutive expression of tetracycline repressor was assayed by Western blot with an anti-tetracycline repressor rabbit polyclonal antibody (MoBiTec, Göttingen, Germany). One clone for each cell line (designed A673/TR and SK-PN-DW/TR, which express the highest levels of tetracycline repressor) was chosen for additional studies.

The target shRNA sequences for EWS/FLI1 and CCK were chosen using the BLOCK-iT RNAi Designer web application (Invitrogen). Two different shRNAs against EWS/FLI1 mRNA were designed: one against the fusion region comprising exon 7 of EWS and exon 6 of FLI1 (shEF) and the other against a sequence localized in the 3' mRNA, and thus common to EWS/FLI1 and FLI1 mRNA (shFLI1). The specific shRNA against CCK (shCCK) corresponds to nucleotides 432 to 452 of the CCK mRNA (GenBank accession no. NM_000729). Oligonucleotide sequences were shEF forward, 5'-CACCGCAGCAGAACCCTTCTTATGACGAATCATAAGAAGGGTTCTGCTGC-3'; shEF reverse, 5'-AAAAGCAGCAGAACCCTTCTTATGATTCGTCATAAGAAGGGTTCTGCTGC-3'; shFLI1 forward, 5'-CACCGGGCACAAACGATCAGTAAGACGAATCTTACTGATCGTTTGTGCCC-3'; shFLI1 reverse, 5'-AAAAGGGCACAAACGATCAGTAAGATTCGTCTTACTGATCGTTTGTGCCC-3'; shCCK forward, 5'-CACCGGACGAATGTCCATCGTTAAGCGAACTTAACGATGGACATTCGTCC-3'; shCCK reverse, 5'-AAAAGGACGAATGTCCATCGTTAAGTTCGCTTAACGATGGACATTCGTCC-3'. Oligonucleotides were annealed and inserted into the pENTR-BLOCK-iT plasmid. After sequence verification, the H1/shRNA cassette was transferred by recombination to the pLenti4-BLOCK-iT plasmid. A673/TR and SK-PN-DW/TR cells were then infected with lentiviruses containing the pLenti4-shRNAs and selected with zeocin (100 µg/mL). After selection, cells were stimulated with doxycycline (1 µg/mL) and mRNA knockdown was assayed by quantitative reverse transcription-PCR (RT-PCR) using specific TaqMan probes and primers. Polyclonal populations and clones displaying the higher levels of mRNA knockdown were chosen for additional studies.

Multiplex real-time quantitative RT-PCR. Total RNA from cells and tumor specimens was extracted with TRI reagent solution. First-strand cDNA was synthesized from 1 µg of total RNA in a 20-µL reaction buffer containing 1x reverse transcriptase, 200 µmol/L of each deoxynucleotide triphosphate, 10 mmol/L DTT, 2.5 µmol/L random hexamers, 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen), and 40 units of RNasin (Promega, Madison, WI). Reaction was incubated at 42°C for 30 min and then at 95° for 5 min. Multiplex real-time quantitative PCR was done from 1.6 µL of reverse transcriptase diluted in a 20-µL reaction containing 1x PCR buffer, 200 µmol/L of each deoxynucleotide triphosphate, 4 mmol/L MgCl2, 0.2 µmol/L of primers, 0.1 µmol/L TaqMan probes, and 0.5 unit of Taq DNA polymerase (Biotools, Madrid, Spain). Reactions were run on a RotorGene 2000 (Corbett Research, Inc., Mortlake, Australia). Cycling conditions consisted of a 5-min denaturation step followed by 35 cycles of denaturation at 95°C for 15 s and an annealing and extension step at 60°C for 50 s. The sequences of the primers and TaqMan probes were, for TATA-binding protein (TBP; used as a reference gene), TBP-F, 5'-GAACATCATGGATCAGAACAACAG-3'; TBP-R, 5'-ATTGGTGTTCTGAATAGGCTGTG-3'; and TBP TaqMan probe, 5'-FAM-CTGCCACCTTACGCTCAGGGCTTGG-TAMRA-3'; and for EWS/FLI1, EWS/FLI1-F, 5'-AGCCAAGCTCCAAGTCAATATAG-3'; EWS/FLI1-R, 5'-GGTTGAGCAGCTTTCGACTG-3'; and EWS/FLI1 TaqMan probe, 5'-TET-AACAGAGCAGCAGCTACGGGCAGCA-TAMRA-3'. For determination of CCK mRNA levels, we used a commercially available TaqMan probe mix (Assay-on-Demand, Applied Biosystems, Foster City, CA). Cycle threshold (Ct) values for EWS/FLI1, CCK, and TBP were calculated using the RotorGene Software and exported to Excel spreadsheets for additional analysis. Relative EWS/FLI1 and CCK expression was calculated as 2–{Delta}Ct, where {Delta}Ct = CtEWS/FLI1 or CCK – CtTBP.

Detection of CCK receptors by RT-PCR. First-strand cDNA was carried out as described above. PCR was done from 3 µL of reverse transcriptase reaction diluted in a 25-µL reaction containing 1x PCR buffer, 200 µmol/L of each deoxynucleotide triphosphate, 2 mmol/L MgCl2, 0.2 µmol/L of primers, and 0.5 unit of Taq DNA polymerase (Biotools). Cycling conditions consisted of a 2-min denaturation step followed by 35 cycles of denaturation at 95°C for 60 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s. Amplicons were visualized by ethidium bromide staining after agarose electrophoresis. The sequences of the primers used to detect the CCK receptors were, for CCK-1 receptor, CCK-1F, 5'-TGAACTCGGGCTCGAAAATGA-3', and CCK-1R, 5'-CATGAAGTAGGTGGTGGTCTT-3'; for CCK-2 receptor, CCK-2F, 5'-ATCTCTCGCGAGCTCTACTTA-3', and CCK-2R, 5'-CAGCAAGTGAATGAAGGAGAT-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control to ensure that similar amounts of RNA were analyzed in each sample. The sequences of the primers specific for GAPDH were GAPDH-F, 5'-ATGGCACCGTCAAGGCTGAG-3', and GAPDH-R, 5'-AGACCACCTGGTGCTCAGTG-3'. PCR products were sequenced to confirm its identity.

CCK RIA. CCK peptide secreted in culture medium was quantified in a commercial CCK RIA (EURIA-CCK, Euro-Diagnostica AB, Medeon, Sweden). Cells were plated at a density of 300,000 per 100-mm plate and allowed to attach for 48 h. Then, cells were incubated in the absence or presence of doxycycline (1 µg/mL) to induce the expression of the corresponding small interfering RNA (siRNA). After 24 h, the medium was removed and cells were cultured with fresh medium with or without doxycycline. After 48 h of additional incubation, conditioned medium (4 mL) was briefly centrifuged to remove cellular debris and extracted in two volumes of ethanol (96%, v/v). After centrifugation at 1,700 x g for 15 min, supernatants were dried in a Speed-Vac, reconstituted to the initial volume (SK-PN-DW cells) or to 1/10 of the initial volume (A673 cells), and assayed in the CCK RIA.

Cell growth assays. Cells were plated at a density of 25,000 per well onto six-well plates and allowed to attach for 24 to 48 h. Where indicated, cells were incubated in the presence of doxycycline (1 µg/mL) from 0 to 6 days. Cell growth was determined by crystal violet staining. DNA synthesis was determined by bromodeoxyuridine (BrdUrd) incorporation into DNA (Cell proliferation ELISA, Roche Applied Science).

Tumor formation assay in nude mice. Tumor cells were washed twice in PBS and resuspended at a density of 5 x 107/mL. Cell suspension was injected (0.1 mL) s.c. into the left flanks of 6-week-old male BALB/c nu/nu mice (Harlan Ibérica, Barcelona, Spain). The animals were kept under pathogen-free conditions and observed daily for the visual appearance of tumors at injection sites. The tumor volume was measured every 2 to 3 days and calculated with the formula L x W x {pi} / 6, where L is the length and W is the width of the tumors. When indicated, doxycycline was given by oral route in natural mineral water at a concentration of 1 mg/mL starting from the same day (experiment 1) or from 3 days before tumor cell injection (experiment 2). When tumor volume reached 1.4 cm3, mice were sacrificed and tumors were removed. A part of the tumor was snap-frozen in liquid nitrogen for determination of CCK and EWS/FLI1 mRNA levels by quantitative RT-PCR and another part was fixed in formalin for immunohistochemical analysis. Animal experiments were done in accordance with institutional guidelines.

Immunohistochemistry. Antigen retrieval on previously dewaxed formalin-fixed, paraffin-embedded sections (3 µm) was done by heating in a microwave oven in Tris-EDTA buffer. After blocking endogenous peroxidase with 0.3% H2O2, sections were incubated at 37°C for 30 min with prediluted monoclonal anti–Ki-67 antibody (clone MIB-1; DAKO, Glostrup, Denmark). Staining was done with the EnVision Dual system (DAKO). The percentage of cells with nuclear staining was evaluated by two pathologists (I.G.M. and D.A.) blinded to clinicopathologic and molecular data.

Statistical analysis. For a single comparison of two groups, two-tailed Student's t test was used. Two-way ANOVA using the Student-Newman-Keuls method was used for comparison of tumor size in control and treated mouse groups. Differences in the appearance of tumors were analyzed by the log-rank test. For all analyses, the level of significance was set at P < 0.05. All statistical calculations were done using the GraphPad Prism statistical software version 4.0 (GraphPad Software, San Diego, CA). Data are presented as mean ± SE.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
In an attempt to identify new targets of the EWS/FLI1 oncoprotein, we analyzed the gene expression profile induced by EWS/FLI1 in HeLa cells. We stably transfected HeLa TeT-On cells, which express high levels of the reverse tetracycline-controlled transactivator, with a tetracycline-responsive expression vector encoding the EWS/FLI1 type 1 cDNA (HeLa TeT-E/F) or with empty vector (HeLa pTET) as a control. In this manner, EWS/FLI1 expression may be induced in HeLa TeT-E/F cells by tetracycline, or its analogue doxycycline, in a dose- and time-dependent fashion (19). RNA from HeLa pTeT and HeLa TeT-E/F cells was isolated from unstimulated cells and after 72 h of stimulation with doxycycline from two independent experiments, and then labeled and hybridized to CodeLink Uniset I Human 10K microarrays containing probes to ~10,000 well-annotated genes. Genes up-regulated at least twice in HeLa TeT-E/F cells on doxycycline stimulation in relation to unstimulated cells and HeLa pTeT control cells, in both experiments, were considered to be specifically regulated by EWS/FLI1. These genes are shown in Table 1 . The FLI1 probe present in the microarray, which maps at the 3' region of FLI1 mRNA and thus also recognizes the EWS/FLI1 mRNA, served as an internal control in these experiments. As expected, FLI1 probe displayed one of the largest increments on induction of EWS/FLI1 expression in HeLa TeT-E/F cells (Table 1).


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  		Table 1. Genes up-regulated by EWS/FLI1 in HeLa TeT-On cells

 
Biologically relevant EWS/FLI1 target genes should also be overexpressed in Ewing tumors. To this purpose, we analyzed the gene expression profile of Ewing tumors (n = 10) and compared it with two unrelated pediatric malignancies, rhabdomyosarcoma (n = 10) and neuroblastoma (n = 7; Fig. 1A ). Tumor RNA was labeled and hybridized to CodeLink Human 20K oligonucleotide microarrays, containing probes to ~20,000 genes, including all the probes already present in the Uniset I Human 10K microarray previously used. We then compared the median of the gene expression for each gene in the Ewing tumor group to the median obtained in the neuroblastoma and rhabdomyosarcoma group. Genes in which the median ratio between groups was >5 and reached a statistical significance of P < 0.005 according to Student's t test with Bonferroni correction were considered to be overexpressed in Ewing tumors. Figure 1A shows the list of these highly specific Ewing genes ranked by the median expression ratio. In addition to newly identified genes with high expression in Ewing tumors, we found several genes known to be highly expressed in Ewing tumors [e.g., neuropeptide Y receptor 1 (NPYR1; ref. 20), six transmemebrane epithelial antigen of the prostate (STEAP; ref. 21), caveolin 1 (CAV1; refs. 22–24), follicular lymphoma variant translocation (FVT1; refs. 22–25), Janus kinase 1 (JAK1; ref. 22–26), inhibitor of DNA binding 2 (ID2; refs. 27, 28), and CCK (29)].


Figure 1
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  		Fig. 1. CCK is overexpressed in Ewing tumors and derived cell lines. A, RNA from Ewing tumors (ET; n = 10), rhabdomyosarcoma (RMS; n = 10), and neuroblastoma (NB; n = 7) was labeled and hybridized to CodeLink Human 20K microarrays. Figure shows genes overexpressed in Ewing tumors as compared with rhabdomyosarcoma and neuroblastoma in which the median ratio of Ewing tumors versus rhabdomyosarcoma and neuroblastoma was >5 and Student's t test with Bonferroni correction between groups reached a P < 0.005. Genes are ranked according median ratio. Right, median ratio and P values from genes with ratio >20. Boxed genes, genes also up-regulated by EWS/FLI1 in HeLa TeT-On cells (Table 1). B, CCK mRNA levels were determined by real-time quantitative RT-PCR in 21 Ewing tumors, 7 Ewing cell lines, and 19 normal tissues. Relative expression of CCK mRNA versus the control gene TBP; representative of two independent experiments done in duplicate (columns, mean; bars, SE). Gray columns, median expression values in Ewing tumors and cell lines. The values of small intestine and fetal brain, which express the highest levels of CCK mRNA among the 19 normal tissues tested, and the median value of the remaining 17 normal tissues (gray column) are shown. Bottom, types of EWS/ETS fusions (exon EWS/exon ETS) present in the Ewing tumors and cell lines. The levels of CCK mRNA in Ewing tumors were close to those observed in small intestine and fetal brain and, on average, 100-fold higher than those detected in the remaining normal tissues.

 
We then looked for genes induced by EWS/FLI1 in HeLa TeT-E/F and also overexpressed in Ewing tumors. Three genes satisfy both criteria: CCK; nuclear receptor subfamily 0, group B, member 1 (NR0B1, also named DAX1), which we have recently characterized as a EWS/FLI1 specific target gene in Ewing tumors (19); and fibulin-6 (FIBL-6; Table 1; Fig. 1). Here, we focus our attention on CCK, a neuroendocrine peptide involved in a wide range of biological functions including regulation of cell growth (18).

As shown in Fig. 1A, CCK ranked at the top of the Ewing specific gene list, indicating that CCK is one of the genes more representative of Ewing tumors, at least when compared with other pediatrics tumors such as rhabdomyosarcoma and neuroblastoma. To evaluate the abundance of CCK in relation to normal tissues, we analyzed the levels of CCK mRNA in a group of Ewing tumors (n = 21), Ewing cell lines (n = 7), and 19 human normal tissues by real-time quantitative RT-PCR. As shown in Fig. 1B, levels of CCK mRNA in both tumors and Ewing cell lines were very similar to those observed in small intestine and fetal brain, which were the normal tissues that expressed the highest levels of CCK mRNA. Levels of CCK mRNA in Ewing tumors and Ewing cell lines were, on average, 100-fold higher than in the rest of normal tissues. These results confirm that CCK is a gene highly expressed in Ewing tumors.

EWS/FLI1 and FLI1 share identical DNA binding domains, and thus they recognize the same sequences in the DNA (13), although several articles have shown that EWS/FLI1 and FLI1 have dissimilar effects on gene expression (14, 19, 28, 30–32). To analyze if FLI1 had the same or different effects on CCK expression than EWS/FLI1, we used HeLa TeT-On cells stably transfected with a tetracycline-responsive vector encoding wild-type FLI1 cDNA (HeLa TeT-FLI1). As shown in Fig. 2A , whereas EWS/FLI1 induced CCK expression after 72 h of doxycycline stimulation, thus confirming the results from microarray experiments, FLI1 had no effect. This result indicates that induction of CCK expression is specific for EWS/FLI1 oncoprotein. It has been shown that EWS/FLI1 target genes and EWS/FLI1-mediated effects vary with different cell backgrounds, presumably because it regulates different genes depending on the cell context (33). To analyze if EWS/FLI1 was able to up-regulate CCK in a different cell context, we used 293 EcR cells engineered to express EWS/FLI1 or FLI1 proteins on ponasterone A stimulation (19). As shown in Fig. 2A, CCK mRNA levels were also up-regulated by EWS/FLI1 in 293 cells, whereas FLI1 expression had no effect. Western blot analysis showed that EWS/FLI1 and FLI1 proteins were expressed to comparable levels in both cell systems, indicating that the differences observed between EWS/FLI1 and FLI1 cannot be attributed to differences in their levels of expression (Fig. 2B). Taken together, these results show that CCK is specifically up-regulated by EWS/FLI1 in two different cell contexts.


Figure 2
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  		Fig. 2. CCK is up-regulated by EWS/FLI1, but not by FLI1, in HeLa and 293 cells. A, time course of CCK mRNA expression. CCK and TBP (control gene) expression was analyzed by real-time quantitative RT-PCR from total RNA extracted from HeLa TeT-On (left) and 293 EcR (right) cell lines. White columns, control cells transfected with empty vectors; black columns, cells that express EWS/FLI1 in response to the stimuli; gray columns, cells that express native FLI1 protein. For each time point (0, 24, and 72 h), expression of CCK was normalized to that of TBP. Columns, mean of two independent experiments done in duplicate; bars, SE. *, P < 0.001, versus cells transfected with empty vector. B, time course of EWS/FLI1 and FLI1 induction. HeLa TeT-E/F and TeT-FLI1 (left) and 293 EcR-E/F and EcR-FLI1 (right) cells were stimulated for different periods of time with doxycycline (1 µg/mL) or ponasterone A (2.5 µmol/L), respectively, and then nuclear protein extract was analyzed by Western blot with an anti-FLI1 antibody. The A673 Ewing tumor cell line and the U937 lymphoblastic cell line are included for comparison of EWS/FLI1 and FLI1 protein levels, respectively. DOX, doxycycline; Pon A, ponasterone A.

 
The fact that CCK is highly expressed in Ewing tumors and derived cell lines and that its expression is up-regulated by EWS/FLI1 in two different cellular backgrounds strongly suggests that CCK is a EWS/FLI1-regulated gene. However, to unambiguously identify CCK as a EWS/FLI1 target gene in Ewing tumors, we carried out a EWS/FLI1 knockdown experiment using the RNA interference technology. We first infected the A673 and SK-PN-DW Ewing cell lines with a lentiviral vector encoding the tetracycline repressor, and a clone for each cell line displaying the highest levels of tetracycline repressor (A673/TR and SK-PN-DW/TR) was subsequently infected with a doxycycline-inducible shRNA lentiviral expression vector directed either against the EWS/FLI1 fusion region (A673/TR/shEF) or against a sequence localized in the 3' region of FLI1 mRNA (A673/TR/shFLI1; Fig. 3A ; see Materials and Methods for details). Because Ewing cell lines do not express wild-type FLI1 mRNA (Fig. 3B), the specific shRNA designed against the 3' region of FLI1 mRNA should exclusively knock down the levels of EWS/FLI1 in these cells. A673/TR and SK-PN-DW/TR cells infected with empty vector were used as control (pLenti). As shown in Fig. 3C, induction of EWS/FLI1- or FLI1-specific siRNAs with doxycycline reduced EWS/FLI1 mRNA levels in A673 and SK-PN-DW cells, ranging from 57% in SK-PN-DW/TR/shFLI1 to 95% in SK-PN-DW/TR/shEF. This reduction in EWS/FLI1 mRNA levels correlated with the decrease in the levels of the EWS/FLI1 protein (Fig. 3D). These results indicate that both siRNAs efficiently knocked down EWS/FLI1 mRNA and protein. As shown in Fig. 3C, EWS/FLI1 knockdown operated by both EWS/FLI1- and FLI1-specific siRNAs was associated with a severe reduction in the levels of CCK mRNA (Fig. 3C). No changes in EWS/FLI1 or CCK mRNA levels were observed in A673/TR/pLenti or SK-PN-DW/TR/pLenti control cells incubated with doxycycline (data not shown). We also tested the effect of EWS/FLI1 silencing on the levels of secreted CCK peptide in the culture medium of Ewing cells using a CCK-specific RIA. As shown in Table 2 , in basal conditions, SK-PN-DW cells secreted, on average, 10-fold more CCK peptide than A673 cells. Induction of EWS/FLI1-specific siRNAs with doxycycline significantly reduced the levels of secreted CCK in the culture medium in both Ewing cell lines. These experiments thus unambiguously show that expression and synthesis of CCK is dependent on EWS/FLI1 in Ewing tumor cells.


Figure 3
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  		Fig. 3. Inducible RNA interference against EWS/FLI1 down-regulates CCK expression in Ewing cells. A, localization of shEF and shFLI1 sequences used to knockdown EWS/FLI1 expression in Ewing tumor cells. B, Western blot with an anti-FLI1 antibody showing that native FLI1 is not expressed in Ewing tumor cells, and thus shFLI1 is specific for EWS/FLI1 in these cells. The lymphoblastic cell line U937, which expresses high levels of FLI1 protein, is included for comparison. C, A673/TR/shEF and shFLI1 (left) and SK-PN-DW/TR/shEF and shFLI1 (right) were stimulated with doxycycline to induce the expression of the corresponding shRNAs against EWS/FLI1. After 48 h, total RNA was extracted and levels of EWS/FLI1 and CCK mRNA were quantified by real-time quantitative RT-PCR. Relative expression of EWS/FLI1 (top) or CCK (bottom) mRNA versus the control gene TBP in a duplicate experiment (columns, mean; bars, SE). EWS/FLI1 silencing down-regulates CCK mRNA in both Ewing cell lines, indicating that CCK expression is regulated by EWS/FLI1 in Ewing cells. D, total protein extracts were analyzed by Western blot with an anti-FLI1 antibody to quantify the levels of EWS/FLI1 protein. The blot was stripped and incubated with an anti-tubulin antibody as a control for loading and transferring. Both EWS/FLI1- and FLI1-specific shRNAs efficiently reduced the levels of EWS/FLI1 protein.

 

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  		Table 2. Effect of EWS/FLI1 silencing on CCK peptide production in Ewing cells

 
The fact that CCK is a EWS/FLI1 target gene in Ewing cells and that CCK was particularly abundant in Ewing tumors prompted us to explore the physiologic role of CCK in these tumors. Several studies have shown that CCK and the related peptide gastrin are potent stimulants of cell growth, either on normal cells or on some types of cancers (18, 34–36). In light of this, we explored the possibility that CCK could be involved in the regulation of cell proliferation of Ewing cells. To ascertain this, we used the same A673/TR and SK-PN-DW/TR cells used before, but in this occasion infected with an inducible lentiviral expression vector encoding a shRNA designed against CCK (A673/TR/shCCK or SK-PN-DW/TR/shCCK). Induction of the CCK-specific siRNA with doxycycline dramatically down-regulated the levels of CCK mRNA in both cell lines. CCK mRNA expression was reduced >95% after 24 h of doxycycline stimulation, and inhibition was sustained at least until 72 h later (Fig. 4A ). By contrast, induction of CCK-specific siRNA had no effect on the mRNA levels of the housekeeping control gene TBP, the EWS/FLI1 oncoprotein (Fig. 4A), and the EWS/FLI1 target gene DAX1 (data not shown), indicating that the effect of the CCK siRNA was highly specific for its cognate mRNA. As expected, levels of CCK mRNA remained constant when the control cells A673/TR/pLenti or SK-PN-DW/TR/pLenti were incubated with doxycycline (data not shown). We also quantified the levels of CCK peptide on CCK silencing using the CCK-specific RIA. As shown in Fig. 4B, CCK silencing decreased the levels of secreted CCK by 90% in both Ewing cell lines.


Figure 4
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  		Fig. 4. Inducible RNA interference against CCK inhibits cell growth and proliferation in Ewing cells. A673/TR and SK-PN-DW/TR cells were infected with an inducible lentiviral expression vector encoding a shRNA designed against CCK and stable clones were selected. A, a polyclonal population and two independent clones of A673/TR/shCCK cells and a polyclonal population of SK-PN-DW/TR/shCCK cells were stimulated with doxycycline for different periods of time and levels of EWS/FLI1 and CCK mRNA quantified by real-time quantitative RT-PCR. Relative expression of EWS/FLI1 and CCK mRNA versus the control gene TBP in relation to unstimulated cells (arbitrarily set to 100). Points, mean of a representative experiment done in duplicate; bars, SE. Induction of the CCK-specific shRNA with doxycycline efficiently knocked down CCK mRNA levels in both Ewing cell lines. B, polyclonal A673/TR/shCCK and SK-PN-DW/TR/shCCK cells were stimulated with doxycycline for 72 h and levels of secreted CCK peptide in the culture medium quantified by RIA. Induction of CCK-specific shRNA with doxycycline reduced the levels of CCK peptide in the conditioned medium of both Ewing cell lines (*, P < 0.0001, versus unstimulated cells). C, polyclonal A673/TR/shCCK and SK-PN-DW/TR/shCCK cells were stimulated with doxycycline for different periods of time and cell growth was quantified by crystal violet staining. Representative of two experiments done in triplicate (columns, mean; bars, SE). The same experiment was done with two independent clones with equivalent results. Induction of CCK-specific shRNA significantly reduced cell growth in the Ewing cell line A673 (*, P < 0.05; **, P < 0.0001, versus unstimulated cells). D, control cells (pLenti; white columns) and polyclonal and clonal populations of A673/TR/shCCK and SK-PN-DW/TR/shCCK cells (black columns) were stimulated with doxycycline for 72 h and DNA synthesis was quantified by BrdUrd incorporation into DNA. Representative of two independent experiments done in triplicate (columns, mean; bars, SE). Percentage of BrdUrd incorporation versus unstimulated cells, which was arbitrarily set to 100. Induction of CCK-specific shRNA significantly reduced DNA synthesis in Ewing cells (*, P < 0.0001, versus the control cells stimulated with doxycycline).

 
Because we were able to efficiently knock down the expression and synthesis of CCK in two Ewing tumor cell lines, we next analyzed the effect of CCK down-regulation on cell growth of the A673 and SK-PN-DW Ewing cell lines. Polyclonal A673/TR/shCCK and SK-PN-DW/TR/shCCK cells were grown for 6 days in the absence or presence of doxycycline and cell growth was quantified by crystal violet staining. After 6 days, cells incubated with doxycycline and thus expressing the CCK-specific siRNA showed a nearly 40% reduction in cell growth (P < 0.0001; Fig. 4C). Similar results were obtained with two independent subclones of A673/TR/shCCK and SK-PN-DW/TR/shCCK cells (data not shown). Thus, CCK silencing correlated with a significant reduction in cell growth. This inhibition was specific to CCK knockdown because levels of EWS/FLI1 were not altered by the CCK-specific siRNA (data not shown). Besides a significant reduction in cell number after 6 days in the presence of doxycycline, we observed neither changes in cell appearance nor a significant increase in the number of detached cells floating in growth medium, which could be indicative of the existence of apoptotic processes. Under the microscope, cells expressing the specific CCK siRNA remained attached on dishes and had normal morphology in comparison with control cells, suggesting that CCK silencing inhibits the growth of Ewing cells without apparently inducing cell death. To confirm that apoptosis is not implicated in the reduction of cell growth induced by CCK silencing, we did flow cytometry analysis. We did not observe any sub-G0 population by flow cytometry after 6 days of doxycycline stimulation, confirming that apoptosis is not induced by CCK knockdown (data not shown).

To confirm the effect of CCK knockdown on Ewing cell growth, we quantified the incorporation of BrdUrd into DNA in two independent populations of A673/TR/shCCK cells and in the polyclonal population of SK-PN-DW/TR/shCCK cells. As shown in Fig. 4D, incorporation of BrdUrd into DNA was reduced to nearly 50% and 30% in A673/TR/shCCK and SK-PN-DW/TR/shCCK, respectively, on induction of the CCK-specific siRNA (Fig. 4D). Doxycycline per se had no effect on DNA synthesis because the respective control cell lines (pLenti) incubated with the same dose of doxycycline did not show a significant variation in the rate of BrdUrd incorporation. These data confirm that CCK down-regulation interferes with cell proliferation in Ewing tumor cells, suggesting that CCK is an autocrine/paracrine growth factor for these cells.

To induce its biological functions, CCK binds to two specific receptors belonging to the seven transmembrane receptor superfamily: the CCK1-R and CCK2-R receptors (37). We thus analyzed by RT-PCR if Ewing cell lines and tumors express these receptors. As shown in Fig. 5 , the A673 and SK-PN-DW Ewing cell lines and all Ewing tumors analyzed (n = 10) expressed at least one of these receptors, indicating that the expression of CCK receptors is a frequent finding in Ewing tumors.


Figure 5
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  		Fig. 5. Expression of CCK receptors in Ewing cell lines and tumors. Analysis of CCK receptor mRNAs (CCK1-R and CCK2-R) by RT-PCR in total RNA extracted from the A673 and SK-PN-DW Ewing cell lines and 10 tumors. RNA from small intestine (INTEST) was used as a control of CCK1-R and CCK2-R mRNA expression. RT-PCR for GAPDH was used as a control to ensure that similar amounts of RNA were analyzed in each sample.

 
If CCK acts as an autocrine/paracrine growth factor, then incubation of Ewing cells with an external source of active CCK should be able to rescue the growth inhibition observed on CCK silencing. Thus, we tried to rescue the growth inhibition produced by CCK silencing in SK-PN-DW/TR/shCCK cells with conditioned medium obtained from SK-PN-DW/TR/shCCK cells incubated in the absence of doxycycline, which contains high levels of CCK peptide, as previously shown in Fig. 4B. As shown in Fig. 6 , the conditioned medium was able to significantly rescue (nearly 60%) the growth inhibition produced by CCK silencing in the SK-PN-DW Ewing cell line. Conditioned medium obtained from SK-PN-DW/TR/shCCK cells cultured in the presence of doxycycline, and thus containing lower levels of CCK peptide, was also able to rescue these cells from the effect of CCK silencing, although to a lesser extent (nearly 35% recovery). Note that although CCK levels were reduced ~10-fold on CCK silencing, significant levels of CCK could be detected in the conditioned medium of SK-PN-DW/TR/shCCK cells incubated in the presence of doxycycline (Fig. 4B). Thus, the degree of the rescue correlated with the content of exogenous CCK present in the culture medium. Taken together, these findings indicate that CCK acts as an autocrine/paracrine growth factor in Ewing tumors.


Figure 6
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  		Fig. 6. Conditioned medium containing high levels of CCK peptide rescues SK-PN-DW cells from the growth inhibition produced by CCK silencing. SK-PN-DW/TR/shCCK cells were cultured for 6 d in the absence or presence of doxycycline to down-regulate CCK expression. Cells incubated with doxycycline were cultured with fresh medium (no conditioned); conditioned medium obtained from SK-PN-DW/TR/shCCK cultured in the absence of doxycycline, containing high levels of CCK peptide (conditioned – DOX); or conditioned medium obtained from SK-PN-DW/TR/shCCK cultured in the presence of doxycycline, containing low CCK levels (conditioned + DOX). Cell growth was quantified by crystal violet staining. Representative of two experiments done in triplicate (columns, mean; bars, SE). Incubation of SK-PN-DW/TR/shCCK with medium containing high CCK levels rescues these cells from the growth inhibition produced by CCK silencing.

 
Finally, we used a nude mouse tumor xenograft model to evaluate the effect of CCK silencing on Ewing tumor growth in vivo. We carried out two independent experiments, which are summarized in Table 3 . In the first one, we injected s.c. two groups of animals (n = 5) with either A673/TR/pLenti control cells or polyclonal A673/TR/shCCK cells and administered doxycycline in the drinking water to both groups. Tumors grew significantly slower in mice injected with A673/TR/shCCK cells and treated with doxycycline (and thus expressing the CCK-specific siRNA) in relation to control mice injected with A673/TR/pLenti cells (P < 0.001). Differences in tumor volume reached a 36% reduction between the two groups 23 days after injection of tumor cells. In the second experiment, we kept two groups (n = 10) in the presence or absence of doxycycline 3 days before A673/TR/shCCK polyclonal tumor cells were implanted. As shown in Fig. 7A , tumors grew significantly slower in mice treated with doxycycline (and thus expressing the CCK-specific siRNA) in relation to control mice treated with vehicle (P < 0.001). Differences in tumor volume reached a 40% reduction between the two groups 23 days after injection of tumor cells and tumor appearance in this second experiment was significantly delayed, from 11.2 days to 16.3 days in the absence or presence of doxycycline, respectively (Table 3). As expected, CCK mRNA expression in tumors from animals treated with doxycycline was down-regulated by >95% as compared with control mice, whereas levels of EWS/FLI1 expression were not significantly different between tumors from both groups (Fig. 7B). We also analyzed by immunohistochemistry the expression of the nuclear antigen Ki-67, which is a documented marker of cell proliferation. As shown in Fig. 7C and D, the percentage of Ki-67–positive cells was significantly reduced in tumors from animals treated with doxycycline. Taken together, these data provide evidence that CCK silencing effectively reduces cell proliferation and tumor growth of Ewing cells in vivo.


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  		Table 3. Effect of CCK silencing on growth of tumor xenografts in athymic nude mice

 

Figure 7
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  		Fig. 7. CCK silencing inhibits proliferation and Ewing tumor growth in vivo. Nude mice were injected s.c. with A673/TR/shCCK cells (n = 10) and treated with (+DOX) or without (–DOX) doxycycline by oral route. A, mean tumor volume up to 23 d after injection. Tumors of mice treated with doxycycline significantly grew slower than those of mice treated with vehicle (*, P < 0.0001, two-way ANOVA). B, levels of CCK and EWS/FLI1 mRNA in tumors were quantified by real-time quantitative RT-PCR. Relative expression of CCK and EWS/FLI1 mRNA versus the control gene TBP. Columns, mean of seven (–DOX) and nine (+DOX) tumors done in duplicate; bars, SE. Induction of the CCK-specific shRNA with doxycycline in vivo efficiently knocked down CCK levels in the A673 Ewing cell line (*, P < 0.015). C, Ki-67–positive cells (proliferating cells) were determined by immunohistochemistry in tumor xenografts with an anti–Ki-67 antibody (clone MIB-1). Columns, mean percentage of Ki-67–positive cells in six to eight tumors per group; bars, SE. *, P < 0.02, versus untreated control mice. D, representative micrographs showing the differences in Ki-67 immunostaining between tumors from mice treated with or without doxycycline. CCK silencing by RNA interference inhibits proliferation of Ewing tumor cells in vivo.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Ewing family of tumors are very aggressive pediatric malignancies. Despite dose-intensive treatments, 30% to 40% of patients with localized and 80% of patients with metastatic disease die due to disease progression (1), making necessary innovative therapies for the treatment of these tumors. Ewing tumors are characterized by chromosomal translocations that yield chimeric transcription factors, such as EWS/FLI1, which regulate the expression of specific genes that contribute to the malignant phenotype (reviewed in ref. 3). Thus, the identification of these EWS/FLI1 target genes and of their contribution to Ewing pathogenesis is a requisite to the understanding of the molecular pathology of these tumors and to the identification of new therapeutic targets.

In this work, we have analyzed the gene expression profile induced by EWS/FLI1 in HeLa cells to identify target genes of the EWS/FLI1 oncoprotein. These results were combined with studies done in tumors and Ewing cell lines to identify biologically relevant EWS/FLI1 target genes. The number of genes regulated by EWS/FLI1 in HeLa cells by microarray analysis resulted low when compared with other studies in which the effect of EWS/FLI1 expression on gene expression profile in other heterologous systems was also studied (24, 38). These differences could be a consequence of the technical differences between the platform used, the different methods of data normalization, the different criteria to select the most relevant genes, and mainly the differences among the cellular systems used in each study. In addition, these finding emphasize the limitations of the studies done in heterologous systems and the necessity to compare the results obtained in these systems with those carried out in tumors and related cell lines (e.g., using RNA interference strategies) to identify biologically relevant genes of oncogenic chimeric transcription factors. In our study, only three genes up-regulated by EWS/FLI1 in HeLa cells were considered to be relevant genes on the basis of its overexpression in Ewing tumors: NR0B1, a gene previously identified by us as a biologically relevant target of EWS/FLI1 and recently shown to be required for the oncogenic phenotype mediated by EWS/FLI1 in Ewing tumors (19, 39); FIBL-6, an extracellular member of the immunoglobulin superfamily implicated in hemidesmosomes organization (40); and CCK, a neuroendocrine peptide involved in regulation of cell growth (18). In this work, we focus our attention on CCK.

We show here that EWS/FLI1, but not native FLI1, induces the expression of CCK in HeLa and 293 cells. More importantly, EWS/FLI1 knockdown using two different siRNAs dramatically reduced the levels of CCK mRNA and secreted peptide in two Ewing cell lines, showing that CCK is a EWS/FLI1-regulated gene in Ewing cells. Whether CCK is a direct or an indirect target of the EWS/FLI1 oncoprotein is at the moment unknown. Preliminary results using luciferase reporter assay indicate that a 1-kb fragment of the CCK promoter, which contains two putative ETS-binding sites, is insufficient to mediate the effect of EWS/FLI1 on CCK expression (data not shown). This finding and the fact that CCK levels were up-regulated late in HeLa cells suggest that CCK is probably an indirect target of the EWS/FLI1 oncoprotein, thus emphasizing the necessity to characterize the mechanism that regulates CCK expression and synthesis in Ewing tumor cells.

We have also quantified the levels of CCK in Ewing tumors and derived cell lines and compared them with other pediatric tumors and normal tissues. Our data show that CCK is overexpressed in Ewing tumors, with levels similar to those detected in small intestine and fetal brain (the two normal tissues tested with the highest levels of CCK) and that CCK can be considered as a Ewing specific marker, at least when compared with other pediatric malignancies such as neuroblastoma and rhabdomyosarcoma. Our studies extend those of Friedman et al. (29) and Schneider et al. (41) in which Ewing tumors were shown to express CCK mRNA. In addition, our data are in agreement with those previously reported by Reubi et al. (42), who suggested the use of CCK as a specific marker of Ewing tumors and for monitoring of therapy. The fact that CCK is a specific target gene of the EWS/FLI1 oncoprotein and that Ewing tumors are among the tumors that express the highest levels of CCK prompted us to explore the physiologic role of CCK in these tumors using the RNA interference approach.

RNA interference is a powerful tool for manipulating gene expression in mammalian cells, with potential usefulness as a therapeutic tool (43). We have used a doxycycline-inducible shRNA system to silence CCK mRNA and to ascertain the functional role of CCK expression in Ewing tumor cells. This system has two main advantages: first, once that stable infectants are obtained, experiments are highly reproducible; second, in vivo induction of shRNAs is easily achieved by addition of doxycycline in the drinking water of the animals. Induction of CCK-specific siRNA with doxycycline provoked a >90% reduction in CCK mRNA levels and secreted peptide, which was associated with a significant inhibition of cell growth in the A673 and SK-PN-DW Ewing cell lines expressing the CCK-specific siRNA. Moreover, when A673 cells were injected s.c. in nude mice, tumor growth and cell proliferation, as assayed by Ki-67 immunostaining, were significantly reduced on induction of CCK-specific siRNA. We have also shown that Ewing cells and tumors express CCK receptors and that incubation of the Ewing cell line SK-PN-DW with an external source of active CCK is able to reverse the growth inhibition produced by CCK silencing. Our data thus indicate that CCK is participating in an autocrine/paracrine loop regulating proliferation of Ewing tumor cells.

Nevertheless, it is worth noting that expression of CCK-specific siRNA did not completely block the proliferation of Ewing tumor cells. Note that although we routinely observed a >90% reduction in the levels of CCK mRNA and peptide, still significant amounts of CCK were secreted by SK-PN-DW cells, and probably also in the A673 cells, on induction of CCK-specific siRNA, although in the latter case the amounts of CCK present in culture medium are most likely below the detection limit of the CCK-specific RIA. Thus, it is possible that residual proliferation is presumably due, in part, to the fact that significant amounts of CCK are still secreted by Ewing cells expressing the CCK-specific siRNA. Consequently, a more effective blocking of CCK should have more striking effects on Ewing cell proliferation. Alternatively, the incomplete inhibition observed on induction of CCK-specific shRNA could also imply that CCK is not the only factor involved in the autocrine regulation of Ewing cell proliferation. One or several other growth factors can also participate in the growth regulation of Ewing tumor cells as have been shown for insulin-like growth factor I and platelet-derived growth factor (44–46).

The cure rate for patients with Ewing's sarcoma, particularly those who present with large tumors or metastatic disease, is poor and survival rates have remained stagnant over the past 20 years despite aggressive dose-intensive chemotherapy combined with radiation and surgery. Therefore, novel targeted therapies are necessary in an effort to improve the outcomes of these patients. Our findings suggest that CCK targeting may have therapeutic benefit. Whereas it is unlikely that CCK targeting will be able to completely eliminate Ewing tumors by itself, one can expect that their combination with standard chemotherapy will synergistically increase tumor kill and decrease the development of resistance by interfering with different pathways essential for tumor cell survival. Several studies indicate that the sensitivity of cancer cells to cytotoxic agents may be enhanced by exposure of cells to inhibitors of growth factor actions. For example, several in vitro and in vivo studies have shown the efficiency of treatments combining classic chemotherapeutic drugs and inhibitors of autocrine growth factor pathways such as insulin-like growth factor I (47, 48). Additional studies are thus necessary to ascertain if combination of CCK inhibition with classic chemotherapy agents used in Ewing therapy, such as vincristine, actinomycin D, or ifosfamide, is more effective in producing growth inhibition or cell death.

In summary, we have shown that CCK is a specific target gene of the EWS/FLI1 oncoprotein in Ewing tumor cells. We have also shown that inhibition of CCK expression with RNA interference efficiently inhibits cell proliferation and tumor growth of Ewing tumor cells, indicating that CCK plays an important role in the autocrine regulation of cell proliferation in these tumors. Understanding the mechanism for which CCK regulates proliferation in Ewing cells should contribute to the development of targeted therapies, which are necessary to improve the outcomes of Ewing tumor patients.


   Acknowledgments
 
We thank Dr. E. Lalli for support in proofreading the manuscript and helpful comments and Roberto Martín and Isabel Camino for technical assistance.


   Footnotes
 
Grant support: Ministerio de Educación y Ciencia grant SAF2003-01068; Instituto de Salud Carlos III, Ministerio de Sanidad grants G03/089 and C03/10; Fundación Inocente Inocente; and Fundación Enriqueta Villavecchia. J. Carrillo and E. García are predoctoral fellows from the Ministerio de Educación y Ciencia. N. Agra is a predoctoral fellow from the Fundación General de la U.A.M. J. Alonso is supported by a Ramón y Cajal contract from the Ministerio de Educación y Ciencia.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/18/06; revised 12/ 4/06; accepted 1/10/07.


   References
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 Materials and Methods
 Results
 Discussion
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