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Molecular Cancer Research 4:851-859 (2006)
© 2006 American Association for Cancer Research

Cancer Genes and Genomics

NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma

Michelle Kinsey1, Richard Smith2 and Stephen L. Lessnick1,2,3

1 The Department of Oncological Sciences; 2 The Center for Children, Huntsman Cancer Institute; and 3 The Division of Pediatric Hematology/Oncology, University of Utah, Salt Lake City, Utah

Requests for reprints: Stephen L. Lessnick, Huntsman Cancer Institute, 2000 Circle of Hope, Room 4242, Salt Lake City, UT 84112. Phone: 801-585-9268; Fax: 801-585-5357. E-mail: stephen.lessnick{at}hci.utah.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
A number of solid tumors, such as alveolar rhabdomyosarcoma, synovial sarcoma, and myxoid liposarcoma, are associated with recurrent translocation events that encode fusion proteins. Ewing's sarcoma is a pediatric tumor that serves as a prototype for this tumor class. Ewing's sarcomas usually harbor the (11;22)(q24;q12) translocation. The t(11;22) encodes the EWS/FLI fusion oncoprotein. EWS/FLI functions as an aberrant transcription factor, but the key target genes that are involved in oncogenesis are largely unknown. Although some target genes have been defined, many of these have been identified in heterologous model systems with uncertain relevance to the human disease. To understand the function of EWS/FLI and its targets in a more clinically relevant system, we used retroviral-mediated RNAi to "knock-down" the fusion protein in patient-derived Ewing's sarcoma cell lines. By combining transcriptional profiling data from three of these lines, we identified a conserved transcriptional response to EWS/FLI. The gene that was most reproducibly up-regulated by EWS/FLI was NR0B1. NR0B1 is a developmentally important orphan nuclear receptor with no previously defined role in oncogenesis. We validated NR0B1 as an EWS/FLI-dysregulated gene and confirmed its expression in primary human tumor samples. Functional studies revealed that ongoing NR0B1 expression is required for the transformed phenotype of Ewing's sarcoma. These studies define a new role for NR0B1 in oncogenic transformation and emphasize the utility of analyzing the function of EWS/FLI in Ewing's sarcoma cells. (Mol Cancer Res 2006;4(11):851–7)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
The ETS family of transcription factors is important for the development of a wide variety of malignancies (1). With the recent finding of translocations involving two different ETS family members in prostate cancer, alterations of ETS proteins may be the most common abnormality in human cancer (2). Understanding the mechanisms by which this transcription factor family contributes to oncogenesis may lead to new diagnostic, prognostic, and therapeutic approaches.

Ewing's sarcoma is a highly malignant bone-associated pediatric cancer that serves as an excellent model to understand the role of ETS family members in tumor formation. Most cases of Ewing's sarcoma harbor a recurrent translocation, (11;22)(q24;q12), that encodes the EWS/FLI fusion oncoprotein (3). EWS is a protein of uncertain function, whereas FLI is an ETS family member (3). EWS/FLI is an aberrant transcription factor that requires both the FLI-derived ETS DNA binding domain, and the EWS-derived strong transcriptional activation domain for its oncogenic activity (4-6). The target genes that mediate EWS/FLI function are only beginning to be understood.

The cell of origin of Ewing's sarcoma is unknown, hence, most studies of EWS/FLI have relied on various heterologous cell types with uncertain relevance to the human disease (e.g., refs. 5, 7-10). In addition, it has been difficult to functionally validate putative EWS/FLI targets as being important for the oncogenic phenotype of Ewing's sarcoma because of the lack of a clinically relevant model system to study these proteins. Thus, it is uncertain whether many of the previously identified EWS/FLI targets are involved in the bona fide tumor.

We recently developed an approach to study the function of EWS/FLI in its native cellular context (11). We used retroviral-mediated RNAi to knock-down endogenous EWS/FLI expression in patient-derived Ewing's sarcoma cells. When coupled with microarray analysis, we were able to define the transcriptional response to EWS/FLI in Ewing's sarcoma. Although this was an important next step, one drawback of our earlier work is that it was limited to a single Ewing's sarcoma cell line, A673. Thus, some of the EWS/FLI-regulated genes which we identified are likely to be specific to A673, and not generally applicable.

We now report an analysis of EWS/FLI-regulated genes across three Ewing's sarcoma cell lines. In this way, we have defined a "core" transcriptional response to the fusion protein. Because these genes are dysregulated in all our analyzed cell lines, they are likely to be enriched in targets that are required for tumor formation. Consonant with this expectation, we identified NR0B1 as an EWS/FLI-regulated gene that is required for Ewing's sarcoma oncogenic transformation. NR0B1 is an orphan nuclear receptor that is important for adrenal gland development (12). NR0B1 has not been previously shown to be functionally important for cancer formation. Our results thus define a functionally important target gene for EWS/FLI, show a new function for NR0B1, and provide a link between EWS/FLI and dysregulation of developmental pathways in pediatric cancer.


   Results and Discussion
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 Acknowledgements
 References
 
We analyzed the EWS/FLI-mediated transcriptional profile in two Ewing's sarcoma cell lines that had not been previously evaluated: TC71 and EWS502. Each cell line was infected with a retrovirus expressing a short hairpin RNA that targeted the 3'-untranslated region of EWS/FLI (called EF-2-RNAi), or a negative control retrovirus directed against luciferase (which is not expressed in these cells; called luc-RNAi). Polyclonal pools of infected cells were recovered following puromycin selection and were used for subsequent assays. EWS/FLI was efficiently knocked-down in each cell line (Fig. 1A ). Knock-down of the fusion protein resulted in a significant reduction of tissue culture growth (as measured by 3T5 assay; Fig. 1B). The initial decrease in cell number observed with each retroviral construct was likely due to incomplete drug selection at the start of the growth assays, and not increased cell death due to the RNAi constructs.4


Figure 1
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  		FIGURE 1. EWS/FLI is required for normal growth and transformation in TC71 and EWS502 Ewing's sarcoma cells. A. Western blot analysis reveals decreased levels of EWS/FLI protein in both TC71 and EWS502 cell lines infected with the EF-2-RNAi retroviral construct, as compared with luc-RNAi control–infected cells. Tubulin is shown as a loading control. B. 3T5 growth assays reveal that knock-down of EWS/FLI with the EF-2-RNAi retroviral construct results in diminished growth in both TC71 and EWS502 cell lines, as compared with luc-RNAi control–infected cells. C. Soft agar assays show significantly decreased colony formation, and thus, oncogenic transformation in TC71 and EWS502 cell lines infected with EF-2-RNAi retroviral construct, as compared with luc-RNAi control–infected cells. Columns, mean of duplicate samples; bars, SD.

 
In addition to growth arrest, TC71 and EWS502 cells with EWS/FLI knock-down via the EF-2-RNAi construct also exhibited a significant loss of soft agar colony growth (one measure of oncogenic transformation; Fig. 1C). Overall, results from TC71 and EWS502 Ewing's sarcoma cells contrast with those we observed for A673 cells harboring the EF-2-RNAi construct. In the latter case, cells grow normally in tissue culture, but had lost their oncogenic phenotype, as measured by soft agar and xenograft assays (11). The differences in growth observed for all three cell lines suggests that EWS/FLI contributes different (although not mutually exclusive) functions required for transformation. In the case of TC71 and EWS502, EWS/FLI contributes a proliferative function (Fig. 1B). In A673 cells, EWS/FLI is not required for normal growth in tissue culture, but instead contributes a more limited oncogenic function (11). The molecular mechanisms that underlie these differences are currently unknown.

To define the common transcriptional profile mediated by EWS/FLI in these Ewing's sarcoma cells, we prepared RNA from EWS502 and TC71 cells harboring each of the RNAi constructs, and hybridized them to oligonucleotide microarrays. The complete data set can be found as Supplementary Data 1 online. To minimize the contribution of "off-target" or other nonspecific effects (13), we identified a second construct (designated EF-4-RNAi) that also efficiently knocked-down EWS/FLI expression (data not shown). We prepared cells containing this additional construct and also used them for microarray analysis. After preprocessing the data, genes were sorted based on a two-class distinction (EWS/FLI knock-down versus control RNAi) using the signal-to-noise metric (Fig. 2A ). Permutation testing (14) revealed that there were 2,046 genes dysregulated by EWS/FLI at the P < 0.05 significance level: 1,610 were up-regulated and 436 were down-regulated by the fusion protein. The preprocessed data, the rank-ordered data, and the lists of up-regulated and down-regulated genes can be found as Supplementary Data 2 online. These results are somewhat different from those obtained using A673 cells, in which there were three to four times as many genes down-regulated by the fusion than were up-regulated (11). The reason for this difference is unknown, but emphasizes the importance of analyzing the EWS/FLI-mediated transcriptional profile in more than one cell line.


Figure 2
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  		FIGURE 2. Microarray analysis of EWS/FLI target genes in Ewing's sarcoma cells. A. Heat-map representation of the microarray data. Cells infected with luc-RNAi (luc) are controls, whereas cells infected with either the EF-2-RNAi (labeled EF-2) or EF-4-RNAi (EF-4) have diminished EWS/FLI protein levels. Each column indicates one cell sample, and each row indicates one gene. Red, genes that are relatively highly expressed; blue, genes that are expressed at relatively lower levels. The top 200 up-regulated and down-regulated genes are shown. B. Genes that were up-regulated by EWS/FLI in TC71 and EWS502 cells were compared with the genes that were up-regulated by EWS/FLI in A673 cells by GSEA (top left) and by {chi}2 analysis (top right). See text for details of the statistical analysis. The 34 genes that were up-regulated by EWS/FLI in all three Ewing's sarcoma cell lines are listed in Table 1. C. Genes that were down-regulated by EWS/FLI in TC71 and EWS502 cells were compared with the genes that were down-regulated by EWS/FLI in A673 cells by GSEA (top left) and by {chi}2 analysis (top right). The 29 genes that were down-regulated by EWS/FLI in all three Ewing's sarcoma cell lines are listed in Table 2. D. NR0B1 was the gene that was most reproducibly up-regulated by EWS/FLI in all of the cell types tested. The microarray data for NR0B1 in TC71 and EWS502 cells is shown graphically. There are two probe sets for NR0B1 on the Affymetrix U133 Plus 2.0 array, and the data from each probe set is shown separately. Because two distinct preparations of EWS502 cells were used for the microarray analysis, each one is shown separately to demonstrate the reproducibility of the data for NR0B1. The data is normalized to 100% expression in luc-RNAi infected cells (red columns). Blue columns, NR0B1 expression in cells harboring the EF-2-RNAi or EF-4-RNAi constructs. Columns, mean of duplicate samples; bars, SD. E. Western blot analysis reveals that NR0B1 protein is down-regulated when EWS/FLI is knocked-down with the EF-2-RNAi retroviral construct in TC71, EWS502, and A673 Ewing's sarcoma cells. Tubulin is shown as a loading control.

 
To determine how the data obtained in TC71 and EWS502 cells compared with that obtained in A673 cells, we did a series of statistical analyses. The two data sets were generated using different Affymetrix microarray platforms. To allow for gene-to-gene comparisons, we first mapped all probe sets on both arrays to their UniGene identifiers. Each data set was limited to only those genes present on both platforms. We then used Gene Set Enrichment Analysis (GSEA; ref. 15) for the comparison. Simply stated, GSEA determines whether one list of genes is enriched near the top of a second, rank ordered, gene list (15). When two data sets are well-correlated, one expects such an enrichment. This enrichment is quantified using a normalized running-sum statistic called the normalized enrichment score, or NES, and the maximal and minimal NES (MaxNES and MinNES, respectively) are reported. In our implementation of GSEA, the best possible MaxNES is 100 (indicating perfect correlation), and the worst possible MinNES is –100 (indicating perfect anticorrelation). An empirical P value is determined by shuffling the rank ordered list, and determining the number of times that a MaxNES is obtained that is the same, or greater than, the experimental MaxNES. For inverse correlations, the same procedure was used, except that the number of times that a MinNES is obtained that is equal to, or less than, the experimental MinNES is determined. For additional details of the procedure as applied in this report, please refer to the Supplementary Methods online.

We found a highly significant correlation between genes that were up-regulated by EWS/FLI in both data sets (MaxNES = 34.1; P < 0.0001; Fig. 2B; Table 1 ). There was also a highly significant correlation between genes that were down-regulated by the fusion protein in both data sets (MinNES = –39.6; P < 0.0001; Fig. 2C; Table 2 ). Similar correlations were found when {chi}2 analysis was used instead (Fig. 2B and C; Tables 1 and 2). Despite the difference in growth response to EWS/FLI knock-down between TC71 or EWS502 and A673 cells, the gene expression pattern mediated by EWS/FLI was similar regardless of the Ewing's sarcoma cell line being assessed.


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  		Table 1. Genes That Were Up-Regulated by EWS/FLI in All Three Ewing's Sarcoma Cell Lines

 

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  		Table 2. Genes That Were Down-Regulated by EWS/FLI In All Three Ewing's Sarcoma Cell Lines

 
Genes that are involved in oncogenic transformation mediated by EWS/FLI are likely to be similarly regulated in different Ewing's sarcoma cell lines. To identify these, we compared genes that were up-regulated or down-regulated (at P < 0.05) in TC71, EWS502, and A673 cells. There were 34 genes up-regulated, and 29 genes down-regulated by EWS/FLI in all three Ewing's sarcoma cell lines tested (Fig. 2B and C; Tables 1 and 2).

The gene that was most correlated with EWS/FLI expression across all of the data sets was NR0B1, which encodes the NR0B1 protein (also known as DAX1, Fig. 2B and D; Table 1). NR0B1 is an orphan nuclear receptor that is mutated in adrenal hypoplasia congenita and hypogonadotropic hypogonadism (12). Interestingly, NR0B1 has recently been suggested to be an EWS/FLI target gene, however, the functional relevance of this observation was not reported (16). Because NR0B1 expression most correlated to EWS/FLI knock-down in our system, we analyzed the function of NR0B1 in the development of Ewing's sarcoma.

We first validated the microarray data using Western blot analysis. We assessed NR0B1 protein levels in TC71, EWS502, and A673 cells. We found that NR0B1 protein levels were significantly reduced when EWS/FLI was knocked-down in all of these cells, supporting the hypothesis that NR0B1 is regulated by EWS/FLI in Ewing's sarcoma (Fig. 2E).

To determine the role of NR0B1 in Ewing's sarcoma, we developed a retroviral RNAi construct that targets the 3'-untranslated region of the NR0B1 transcript (designated NR0B1-RNAi). This retrovirus was used to infect TC71, EWS502, and A673 Ewing's sarcoma cells. NR0B1-RNAi efficiently knocked down NR0B1 protein levels, as compared with the luc-RNAi control retrovirus (Fig. 3A ).


Figure 3
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  		FIGURE 3. NR0B1 is necessary for oncogenic transformation in Ewing's sarcoma cells. A. Western blot analysis of TC71, EWS502, and A673 Ewing's sarcoma cells reveals that NR0B1 protein expression is efficiently reduced by the NR0B1-RNAi virus, as compared with the luc-RNAi control virus. Tubulin is shown as a loading control. B. Growth (3T5) assays of TC71, EWS502, and A673 cells infected with combinations of the NR0B1 cDNA and NR0B1-RNAi viruses. TC71 maintains normal growth characteristics, whereas EWS502 and A673 have reduced growth rates when NR0B1 expression is knocked down. The growth phenotype is partially reversed by NR0B1 re-expression. C. Soft agar assays reveal a complete loss of oncogenic transformation (as measured by colony formation) in all three Ewing's sarcoma cell lines infected with the NR0B1-RNAi retrovirus. Transformation is rescued by re-expression of NR0B1 protein from the NR0B1 cDNA retrovirus. Columns, mean of duplicate samples; bars, SD. D. Murine xenograft experiments using A673 Ewing's sarcoma cells expressing a luc-neo fusion protein that were subsequently infected with either the ERG-RNAi or NR0B1-RNAi retroviruses show that NR0B1 is required for tumor formation in immunodeficient mice. Left, bioluminescent imaging data; middle, tumor photon emission; right, tumor size, in which numbers indicate separate tumor injection sites. P values from Student's t tests are also shown.

 
We assessed the role of NR0B1 on the growth characteristics of Ewing's sarcoma cells in tissue culture using 3T5 growth assays. Knock-down of NR0B1 resulted in a significant inhibition of EWS502 and A673 growth, but not TC71 growth (Fig. 3B). These growth patterns are reminiscent of the effects we previously observed with EWS/FLI knock-down (Fig. 1B; ref. 11). Interestingly, TC71 cells required EWS/FLI expression, but not NR0B1 expression, for normal tissue culture growth. This suggests that a different EWS/FLI target gene (or genes) is required for proliferation in tissue culture. In the case of EWS502 and A673, NR0B1 is required for normal growth.

Expression of an NR0B1 cDNA that did not contain the 3'-untranslated region (and was thus immune to the effect of NR0B1-RNAi) partially rescued the growth deficit caused by NR0B1 knock-down in EWS502 and A673 cells (Fig. 3B). Thus, the observed effects were due to NR0B1 loss, and not an off-target or other nonspecific RNAi effect (13). Supraphysiologic expression of NR0B1 seemed to be well-tolerated in all three cell lines, as shown by the essentially unchanged growth rate of cells expressing the control luc-RNAi and the NR0B1 cDNA, as compared with cells harboring the luc-RNAi and an empty cDNA vector (Fig. 3B).

Soft agar assays were done to determine if the expression of NR0B1 is also necessary for the transformed phenotype of Ewing's sarcoma cells. Reduction of NR0B1 levels resulted in a significant reduction of oncogenic transformation in TC71, EWS502, and A673 Ewing's sarcoma cells (Fig. 3C). Importantly, this was also true for TC71 cells, which maintained normal growth in tissue culture following NR0B1 knock-down (Fig. 3B), demonstrating that loss of transformation was not simply a consequence of diminished tissue culture growth. Transformation was rescued following re-expression of NR0B1 using the cDNA described above (Fig. 3C), demonstrating that loss of transformation was due to loss of NR0B1 expression. NR0B1 expression was also required for xenograft formation, because knock-down of the transcript diminished tumor growth in vivo (Fig. 3D). Ongoing NR0B1 expression is therefore required for oncogenic transformation in Ewing's sarcoma cells.

We next assessed the intrinsic oncogenic potential of NR0B1. We introduced the NR0B1 cDNA into NIH3T3 immortalized murine fibroblasts. NIH3T3 cells are transformed by a variety of oncogenes (including Ras and EWS/FLI) and are a classic model system to assess oncogenic function (5, 17). Although NIH3T3 cells infected with the NR0B1 cDNA-containing retrovirus expressed robust levels of NR0B1 protein (data not shown), these cells did not form colonies in soft agar (Fig. 4A ). Hence, NR0B1 does not cause oncogenic transformation in NIH3T3 cells.


Figure 4
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  		FIGURE 4. NR0B1 is not sufficient for oncogenic transformation. A. Soft agar assays show that whereas EWS/FLI expression causes oncogenic transformation of NIH3T3 cells (as measured by colony formation), NR0B1 expression does not. "Empty" MSCV-Hygro and pMSCV-Neo are the control viruses for the adjacent samples. The controls do not contain a cDNA. Columns, mean of duplicate samples; bars, SD. B. Soft agar assays show that whereas EWS/FLI expression rescues the loss of oncogenic transformation caused by the EF-2-RNAi retroviral construct, NR0B1 expression does not. Columns, mean of duplicate samples; bars, SD. C. Soft agar assays show that the expression of both NR0B1 and NKX2.2 are insufficient to rescue oncogenic transformation caused by the EF-2-RNAi retroviral construct. Columns, mean of duplicate samples; bars, SD.

 
To assess the oncogenic potential of NR0B1 in a system related to Ewing's sarcoma, we asked whether NR0B1 could rescue the transformation defect in A673 cells caused by the knock-down of EWS/FLI. A673 cells were first infected with EF-2-RNAi retrovirus, and subsequently infected with the NR0B1 cDNA retrovirus. These double-infected cells did not form colonies in soft agar assays, although the EWS/FLI cDNA did rescue transformation caused by knock-down of the endogenous fusion protein (Fig. 4B). Taken together, we conclude that NR0B1 does not function as an oncogene by itself, but rather is a required downstream target of EWS/FLI in Ewing's sarcoma.

The insufficiency of NR0B1 alone to rescue oncogenic transformation following knock-down of EWS/FLI suggested that other EWS/FLI downstream targets participate in this process. We recently identified NKX2.2 as another EWS/FLI up-regulated gene that is required for oncogenic transformation mediated by the fusion protein (11). We therefore asked whether expression of both downstream targets, NR0B1 and NKX2.2, could rescue the phenotype caused by EWS/FLI knock-down. We introduced the cDNAs for both NR0B1 and NKX2.2 into A673 Ewing's sarcoma cells, and recovered a population of cells that expressed both proteins using a combination of neomycin and hygromycin selection. Control cells were also prepared that contained the "empty-vector" controls for each marker. We then introduced either the EF-2-RNAi, or control luc-RNAi, retroviral vectors. Following additional selection in puromycin, these "triple-infectants" were assessed for oncogenic transformation. We found that expression of both NR0B1 and NKX2.2 was insufficient to rescue transformation (Fig. 4C). These data suggest that, in addition to NR0B1 and NKX2.2, additional EWS/FLI dysregulated targets also contribute to the transformed phenotype of Ewing's sarcoma cells.

If NR0B1 is a clinically relevant EWS/FLI target, it should be expressed in primary patient-derived tumor samples in addition to the cell lines described above. To test this hypothesis, we did reverse transcriptase-PCR assays on total RNA isolated from Ewing's sarcoma tumor samples. We found NR0B1 expression in four of four tumors analyzed, but not in primary human fibroblasts (used as a negative control; Fig. 5A ). NR0B1 expression seems to be specific to Ewing's sarcoma. Western blot analysis of 37 different tumor cell lines revealed that NR0B1 protein was only expressed in Ewing's sarcoma cells, but not in lines derived from non–Ewing's sarcoma tumor types (Fig. 5B).


Figure 5
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  		FIGURE 5. NR0B1 expression is limited to Ewing's sarcoma. A. Expression of NR0B1 in primary Ewing's sarcoma tumor samples. Reverse transcriptase-PCR reveals that NR0B1 RNA is expressed in primary patient Ewing's sarcoma tumor samples. A673 cells are shown as a positive controls, normal human fibroblasts are shown as a comparison, and water was used as a negative reaction control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an amplification control. B. Western blot analysis of the indicated cell lines reveals that NR0B1 is only expressed in Ewing's sarcoma lines. The specific NR0B1 band is the lower band in the samples labeled with a "+" below the lanes, whereas the upper band present in all samples is a nonspecific background band. Tubulin is shown as a loading control.

 
The data presented here shows that NR0B1 is an EWS/FLI-regulated gene that is necessary, but not sufficient, for oncogenic transformation in Ewing's sarcoma cells. Although previous studies have documented NR0B1 expression in some tumor types, such as adrenocortical, breast, and ovarian carcinomas (18-20), a functional role for NR0B1 in cancer development has not been shown. Our findings therefore provide a novel role for this orphan nuclear receptor. Surprisingly, we did not observe NR0B1 expression by Western blot in cell lines derived from those tumor types previously reported to express the protein (Fig. 5B; refs. 18-20). This suggests that NR0B1 expression is not required for the growth of these tumor types in tissue culture.

The mechanism by which NR0B1 participates in cancer development is currently unknown. Prior work suggests that NR0B1 functions as a transcriptional repressor or corepressor (12, 21-25). We found that a large number of genes were down-regulated by EWS/FLI. Future work will be required to determine if NR0B1 is the central mediator of these down-regulatory events.

The ligand for NR0B1, if one exists, is unknown. Therefore, we do not know if NR0B1 functions as a repressor in the presence of ligand. Other nuclear hormone receptors, such as the estrogen receptor, androgen receptor, and retinoic acid receptor, serve as important therapeutic targets in diseases such as breast cancer, prostate cancer, and acute promyelocytic leukemia, respectively (26-28). It is possible that a ligand agonist or antagonist for NR0B1 may alter its function and prevent its participation in Ewing's sarcoma development. Thus, the work presented in this report provides support for evaluating NR0B1 as a potential therapeutic target. An NR0B1 agonist or antagonist may function as an antineoplastic agent for Ewing's sarcoma; such an agent would be most welcome for this devastating pediatric cancer.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
Acknowledgements
 References
 
Constructs and Retroviruses
The EF-2-RNAi, EF-4-RNAi, and luc-RNAi retroviruses were previously described (11). To generate the NR0B1-RNAi retroviral vector, the following oligonucleotides were annealed and cloned downstream of the U6 promoter in the pMKO.1P vector (29):

NR0B1-RNAiF: 5'-CCGGCCACACAAGTGCAGTAGTGTTCAAGAGACACTACTGCACTTGTGTGGTTTTTG-3',
NR0B1-RNAiR: 5'-AATTCAAAAACCACACAAGTGCAGTAGTGTCTCTTGAACACTACTGCACTTGTGTGG-3'.

The coding region of NR0B1 was amplified by reverse transcriptase-PCR and cloned into the retroviral expression vector pMSCV-Neo (Clontech, Mountain View, CA). The coding region of EWS/FLI was cloned into the pMSCV-Hygro vector (Clontech). Retroviral production and infection were done as previously described (7).

Cell Culture
Ewing's sarcoma cell lines were grown as described (7). Following retroviral infection, polyclonal pools of cells were selected and maintained in the appropriate selective medium (2 µg/mL for puromycin, 300 µg/mL for G418, and 100 µg/mL for hygromycin). The day when a control uninfected plate of cells was completely dead was considered to be day 0 for subsequent growth assays. Because the efficiency of infection is relatively high (>50% of cells infected), adequate cells for later assays could usually be obtained following drug selection, even in cases in which the cells are growth-arrested.

Growth assays (3T5 assays) were done by plating 5 x 105 cells on a 10 cm (diameter) tissue culture dish in 10 mL of selective growth medium. Cells were harvested and counted 3 days later, and 5 x 105 cells were replated on a new dish. The process was repeated every 3 days for a total of 15 days. Population doubling was calculated as the ln(cell count at day 3 post-plating/cell count plated) divided by ln(2). The cumulative population doubling was calculated by adding the population doubling at a given 3-day interval to the prior cumulative population doubling up to that time point.

Soft agar assays were done as described (7). Quantitation of agar colony formation was done using the Quantity One software package (Bio-Rad, Hercules, CA). Details regarding the growth of the non–Ewing's sarcoma cell lines used is included in Supplementary Methods online.

Xenograft Imaging
A673 cells were infected with pMMP-LucNeo and selected with G418 (30). They were then infected with either NR0B1-RNAi or ERG-RNAi retroviruses, and selected with puromycin. Following selection, 1 x 106 cells were injected into the flanks of nude mice. Mice were imaged weekly using a Xenogen IVIS 100 imaging system, and used according to the manufacturer's directions. Animal experiments were done following approval from the University of Utah Institutional Animal Care and Use Committee.

Reverse Transcriptase-PCR
Total RNA samples derived from primary patient Ewing's sarcoma tumors were provided by Dr. R.L. Randall (Huntsman Cancer Institute). NR0B1 and glyceraldehyde-3-phosphate dehydrogenase fragments were amplified using a one-step reverse transcriptase-PCR kit (Roche, Pleasanton, CA). Primer sequences are available on request.

Microarray and Statistical Analysis
A complete description of the microarray analysis, GSEA, and {chi}2 analysis is provided as Supplementary Methods online. The complete microarray data set from the TC71 and EWS502 cell lines is provided as Supplementary Data 1 online. The preprocessed data, rank-ordered data set, and up-regulated and down-regulated gene sets are provided as Supplementary Data 2 online. The .cls file required to perform the described analysis in either the GeneCluster 2.0 or GenePattern software packages (Broad Institute, Cambridge, MA) is provided as Supplementary Data 3 online.

Western Blot
Protein lysates were prepared in protein lysis buffer [10 mmol/L Tris (pH 7.6), 100 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS] containing protease inhibitors. Total proteins (75-100 µg) were separated by SDS-PAGE, and transferred to nitrocellulose membranes. Anti-NR0B1 (R&D Biosciences, Minneapolis, MN), anti-FLI (Santa Cruz, Santa Cruz, CA), and anti-{alpha}-tubulin (Santa Cruz) were used for immunodetection.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank R.L. Randall for the Ewing's sarcoma patient RNA, D. Trem, R. Weis, and D. Dunn for assistance with the microarray experiments, M. Lessnick for discussions about modifying the GSEA analysis, and F. Fitzpatrick, D. Fults, D. Jones, S. Kuwada, C.P. Reynolds, T. Triche, D. Wei, and D. Wettstein for cell lines.


   Notes
Top
 Notes
Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: S.L. Lessnick is supported by grant K08 CA96755, the Terri Anna Perine Sarcoma Fund, a Primary Children's Medical Center Foundation Innovative Research Grant, a Hope Street Kids grant, and a Catalyst grant from the University of Utah School of Medicine.

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.

Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

4 M. Kinsey, unpublished observations. Back

Received 4/ 3/06; revised 8/23/06; accepted 9/19/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 

  1. Seth A, Watson DK. ETS transcription factors and their emerging roles in human cancer. Eur J Cancer 2005;41:2462–78.[Medline]
  2. Tomlins SA, Rhodes DR, Perner S, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005;310:644–8.[Abstract/Free Full Text]
  3. Dellatre O, Zucman J, Plougastel B, et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 1992;359:162–5.[CrossRef][Medline]
  4. May WA, Lessnick SL, Braun BS, et al. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol 1993;13:7393–8.[Abstract/Free Full Text]
  5. May WA, Gishizky ML, Lessnick SL, et al. Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A 1993;90:5752–6.[Abstract/Free Full Text]
  6. Lessnick SL, Braun BS, Denny CT, May WA. Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. Oncogene 1995;10:423–31.[Medline]
  7. Lessnick SL, Dacwag CS, Golub TR. The Ewing's sarcoma oncoprotein EWS/FLI induces a p53-dependent growth arrest in primary human fibroblasts. Cancer Cell 2002;1:393–401.[CrossRef][Medline]
  8. Deneen B, Denny CT. Loss of p16 pathways stabilizes EWS/FLI1 expression and complements EWS/FLI1 mediated transformation. Oncogene 2001;20:6731–41.[CrossRef][Medline]
  9. Hu-Lieskovan S, Zhang J, Wu L, Shimada H, Schofield DE, Triche TJ. EWS-FLI1 fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing's family of tumors. Cancer Res 2005;65:4633–44.[Abstract/Free Full Text]
  10. Dauphinot L, De Oliveira C, Melot T, et al. Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2 and c-Myc expression. Oncogene 2001;20:3258–65.[CrossRef][Medline]
  11. Smith R, Owen LA, Trem DJ, et al. Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. Cancer Cell 2006;9:405–16.[CrossRef][Medline]
  12. Zanaria E, Muscatelli F, Bardoni B, et al. An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 1994;372:635–41.[CrossRef][Medline]
  13. Jackson AL, Bartz SR, Schelter J, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003;21:635–7.[CrossRef][Medline]
  14. Golub TR, Slonim DK, Tamayo P, et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999;286:531–7.[Abstract/Free Full Text]
  15. Mootha VK, Lindgren CM, Eriksson KF, et al. PGC-1{alpha}-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 2003;34:267–73.[CrossRef][Medline]
  16. Mendiola M, Carrillo J, Garcia E, et al. The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors. Int J Cancer 2005;118:1381–9.
  17. Shih C, Weinberg RA. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell 1982;29:161–9.[CrossRef][Medline]
  18. Abd-Elaziz M, Akahira J, Moriya T, Suzuki T, Yaegashi N, Sasano H. Nuclear receptor DAX-1 in human common epithelial ovarian carcinoma: an independent prognostic factor of clinical outcome. Cancer Sci 2003;94:980–5.[Medline]
  19. Reincke M, Beuschlein F, Lalli E, et al. DAX-1 expression in human adrenocortical neoplasms: implications for steroidogenesis. J Clin Endocrinol Metab 1998;83:2597–600.[Abstract/Free Full Text]
  20. Conde I, Alfaro JM, Fraile B, Ruiz A, Paniagua R, Arenas MI. DAX-1 expression in human breast cancer: comparison with estrogen receptors ER-{alpha}, ER-ß and androgen receptor status. Breast Cancer Res 2004;6:R140–8.[CrossRef][Medline]
  21. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P. DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 1997;390:311–5.[CrossRef][Medline]
  22. Ito M, Yu R, Jameson JL. DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 1997;17:1476–83.[Abstract]
  23. Zhang H, Thomsen JS, Johansson L, Gustafsson JA, Treuter E. DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors. J Biol Chem 2000;275:39855–9.[Abstract/Free Full Text]
  24. Crawford PA, Dorn C, Sadovsky Y, Milbrandt J. Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1. Mol Cell Biol 1998;18:2949–56.[Abstract/Free Full Text]
  25. Altincicek B, Tenbaum SP, Dressel U, Thormeyer D, Renkawitz R, Baniahmad A. Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita. J Biol Chem 2000;275:7662–7.[Abstract/Free Full Text]
  26. Baselga J, Norton L. Focus on breast cancer. Cancer Cell 2002;1:319–22.[CrossRef][Medline]
  27. Isaacs W, De Marzo A, Nelson WG. Focus on prostate cancer. Cancer Cell 2002;2:113–6.[CrossRef][Medline]
  28. Parmar S, Tallman MS. Acute promyelocytic leukaemia: a review. Expert Opin Pharmacother 2003;4:1379–92.[Medline]
  29. Masutomi K, Yu EY, Khurts S, et al. Telomerase maintains telomere structure in normal human cells. Cell 2003;114:241–53.[CrossRef][Medline]
  30. Rubin JB, Kung AL, Klein RS, et al. A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors. Proc Natl Acad Sci U S A 2003;100:13513–8.[Abstract/Free Full Text]



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