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Clinical Cancer Research Vol. 10, 1344-1353, February 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Vascular Endothelial Growth Factor Expression is Up-Regulated by EWS-ETS Oncoproteins and Sp1 and May Represent an Independent Predictor of Survival in Ewing’s Sarcoma

Bruno Fuchs1, Carrie Y. Inwards2 and Ralf Janknecht1

Departments of1 Biochemistry and Molecular Biology and 2 Surgical Pathology, Mayo Clinic, Rochester, Minnesota


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Tumor markers ideally allow monitoring and prediction of disease progression. In Ewing’s sarcoma, a devastating childhood cancer, only a few reliable prognostic markers have been identified. To this end, we analyzed the expression of four tumor-promoting proteins, cyclin D1, HER2/Neu, Mdm2, and vascular endothelial growth factor (VEGF), in Ewing’s sarcoma.

Experimental Design and Results: Thirty-one tissue samples from patients with Ewing’s sarcoma were stained with antibodies against cyclin D1, HER2/Neu, Mdm2, or VEGF. Whereas no significant expression of HER2/Neu and Mdm2 was detected, positive cyclin D1 and VEGF staining was observed in 42% and 55% of all tumors, respectively. Importantly, VEGF expression was found to be an independent negative predictor of survival in Ewing’s sarcoma patients, whereas cyclin D1 expression did not correlate with survival in these patients. Consistently, the Ewing’s sarcoma-specific EWS-ETS oncoproteins were capable of activating both the cyclin D1 and VEGF promoters in transient transfections of tissue culture cells. Furthermore, this activation was enhanced by coexpression of the Sp1 transcription factor. Using a mammalian two-hybrid system, some evidence was obtained that this may involve a physical interaction between EWS-ETS and Sp1 proteins.

Conclusions: Our data reveal that VEGF may serve as a prognostic marker in Ewing’s sarcoma patients and provide a molecular mechanism by which VEGF and cyclin D1 expression is up-regulated in approximately half of all Ewing’s sarcomas.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ewing’s sarcoma is a devastating disease that affects predominantly children and adolescents. With the detection of the first Ewing’s sarcoma-associated fusion protein EWS-Fli1 more than a decade ago (1) , great hope was nourished that this finding may lead to a simple mechanistic explanation of the pathogenesis of this tumor. However, recent years showed that the underlying pathway to cancer formation is more complicated. Therefore, there is a dire need to further study disease mechanisms and to identify potential markers that may assist to combat Ewing’s sarcoma.

The histopathological progression from preneoplasia to cancer goes along with mutations in gene sequences and alterations in protein expression levels. Molecular changes that occur very frequently or even exclusively in a specific malignant cell can be used as markers of cancer. These molecular markers may allow detection of early stages of cancer, monitoring disease progression and efficacy of treatment responses. However, the usefulness of a molecular marker cannot be fully evaluated until its sensitivity, specificity, and correlation with patient survival have been rigorously investigated (2) .

High-throughput screening technologies that allow the simultaneous analysis of expression patterns of several genes and proteins have been used to search for cancer-associated molecules (3 , 4) . With respect to Ewing’s sarcoma, several different approaches were used to identify tumor-specifically expressed genes, and hundreds of genes either being up- or down-regulated were reported (5, 6, 7) ; however, validation of these gene expression changes as molecular markers of Ewing’s sarcoma has presently not been followed up by studying whether corresponding changes in respective proteins levels occur in Ewing’s sarcoma tissue specimens, and whether they are of prognostic value. As an alternative approach, we elected to study a limited number of proteins that are known key players in tumorigenesis but have been poorly characterized in Ewing’s sarcoma, namely, Mdm2, HER2/Neu, cyclin D1, and vascular endothelial growth factor (VEGF).

Mdm2 is an important regulator of the tumor suppressor p53 (8) . Overexpression of Mdm2, as often observed in tumors, leads to increased degradation of p53 and therefore promotes survival of tumor cells. The oncoprotein HER2/Neu belongs to the family of receptor tyrosine kinases, is overexpressed in several cancers (in particular, breast and ovarian cancer, where it correlates with poor prognosis), and has become an attractive target for pharmacological intervention (9 , 10) . Cyclin D1 is a critical regulator of progression through the G1 phase of the cell cycle, and its overexpression is implicated in the development of several cancers (8 , 11) . Finally, VEGF is the key stimulator of endothelial cell growth (12 , 13) and thereby mediator of angiogenesis that is required for efficient tumor proliferation (13, 14, 15, 16, 17, 18, 19) .

In this report, we immunohistochemically analyzed the expression of Mdm2, HER2/Neu, cyclin D1, and VEGF in 31 Ewing’s sarcoma specimens and correlated their overexpression to patient survival. Furthermore, we analyzed how EWS-ETS oncoproteins that are the common denominator of Ewing’s sarcomas (20 , 21) are able to cause enhanced cyclin D1 and VEGF expression by activating the respective gene promoters.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
Thirty-one consecutive patients who were diagnosed with Ewing’s sarcoma at the authors’ institution between 1979 and 1982 were included in this study to ensure long-term follow-up with reliable assessment of disease outcome. The study was approved by the institutional review board.

Immunostaining.
Paraffin-embedded tumor tissues from surgical specimens (before chemotherapy) were cut in 5 µm-thick sections and placed on superfrost charged slides. Immunohistochemistry was performed essentially as described previously (22) . In brief, the slides were deparaffinized in xylene (2 x 5 min), rehydrated in absolute ethanol and a series of ethanol/water mixtures, and rinsed with tap water. Endogenous peroxidase activity was blocked by using 0.3% H2O2 in methanol (1:1, v/v). After a tap water rinse, sections were placed into 1 mM preheated EDTA (pH 8) and then steamed for 30 min. After cooling in buffered 1 mM EDTA (pH 8) for 5 min, the sections were rinsed in tap water and placed in PBS (pH 7.4). Sections were blocked for 5 min with DAKO Peroxidase Block (DAKO Cytomation, Carpinteria, CA) and then incubated with anti-VEGF antibody (NeoMarkers; Labvision) at a 1:10 dilution for 30 min. The DAKO mouse EnVision+ HRP System/AEC+ and the DAKO Autostainer were used for detection. Sections were counterstained with light hematoxylin and then mounted with a coverslip. The same protocol was used for cyclin D1 immunostaining, with the anti-cyclin D1 antibody (DAKO) at a dilution of 1:75 and using the DAKO mouse EnVision+/DAB+ chromogen as the detection system. The conditions for Mdm2 were the same as those for cyclin D1, except that the anti-Mdm2 antibody (DAKO) was used at 1:100 dilution. For staining of HER2/Neu, the DAKO Hercept Test Kit was used.

Control stains for tissue quality and to assure diagnosis were performed with anti-MIC2 antibodies (1:50 dilution; DAKO) and basic routine H&E staining (modified Schmidt’s hematoxylin). Furthermore, the following tissues served as positive controls for the immunohistochemical stainings: placenta (VEGF); breast carcinoma (cyclin D1 and HER2/Neu); and colon cancer (Mdm2). Immunostainings were graded as positive (intense staining) or negative (absent or only faint staining) as represented in Fig. 1Citation .



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  		Fig. 1. Immunostaining for cyclin D1 and vascular endothelial growth factor in Ewing’s sarcoma tissue samples. Two representative patient samples are shown: one staining negative (top row); and one staining positive (bottom row). Control stains with anti-MIC2 antibodies and H&E confirmed the diagnosis of Ewing’s sarcoma.

 
Statistical Analysis.
Data were analyzed with the use of the Fisher’s exact test and a Pearson {chi}2 test for qualitative variables. When quantitative values were evaluated, ANOVA was performed to determine significance. Gender, age at diagnosis, presence or absence of metastasis at diagnosis, anatomical sites, types of treatment, overexpression of cyclin D1 and VEGF, and length of follow-up, which were all adjusted for the disease status at follow-up, were univariately assessed. Factors that showed a significant univariate association with survival were placed into a multivariate Cox proportional hazards model. A significance level of 0.05 was used for all tests. For survival analysis, the method of Kaplan and Meier was used.

Plasmids.
EWS-ER81, EWS-CTD, and EWS-NTD were cloned into pCS3+-6Myc. The Sp1 expression plasmid, Sp1-pEVRF-2, was a gift from Dr. T. C. Spelsberg, and VEGF-Kpn (-2274 to +379)-pGL2-Basic (23) , from which all other VEGF promoter constructs were derived, was kindly provided by Dr. G. Semenza. All promoter constructs were created using PCR, cloned into pGL2-Basic, and verified by DNA sequencing.

Reporter Gene Assays.
Cells were grown overnight on 6-cm dishes and transfected using the calcium phosphate coprecipitation method. Thirty-six h later, cell extracts were prepared, and luciferase activity was then measured in a Berthold lumat as described previously (24) . All experiments were performed at least three times.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Mdm2, HER2/Neu, Cyclin D1, and VEGF in Ewing’s Sarcoma Specimens.
The Ewing’s sarcoma tissue samples analyzed in this study were gathered from open biopsies before initiation of chemotherapy. The patient group encompassed 10 women and 21 men, and the average age was 16.9 years (range, 6–57 years). In 18 patients, the disease was localized at the time of presentation, whereas 13 patients presented with metastasis. Twenty-three patients underwent chemotherapy with the same protocol. Thirteen patients had the lesion in the axial skeleton, and 18 patients had the lesion in the appendicular skeleton. These patients had a mean follow-up of 83 months (range, 3–249 months). At the final follow-up, 11 patients were alive, whereas 20 patients had died of disease.

All of the tumors included in this study showed the typical histological features of Ewing’s sarcoma. They contained round-to-oval nuclei with little variability in size and shape. The cells contained scant, slightly granular eosinophilic cytoplasm, and the cell borders were indistinct. In addition, all of the tumors showed positive immunoreactivity with antibodies to CD99/MIC2, an established diagnostic marker for Ewing’s sarcoma (25 , 26) . Immunohistochemical analyses revealed that all 31 tissue samples stained negative for HER2/Neu and Mdm2 (data not shown). In contrast, 13 tissue samples (42%) stained positive for cyclin D1, and 17 tissue samples (55%) stained positive for VEGF (Fig. 1)Citation .

Survival Analysis.
The overall survival of the 31 patients included in this study was 81%, 55%, 45%, and 45% at 1 year, 3 years, 5 years, and 10 years, respectively (Fig. 2A)Citation . These findings are comparable with those of other studies on Ewing’s sarcoma patients (27 , 28) . One of the most accepted negative prognostic parameters of survival for Ewing’s sarcoma patients is the presence of metastatic disease at presentation (29) . Indeed, there is a significant (P = 0.022) difference in survival between patients who presented with localized versus metastatic disease in our study population (Fig. 2B)Citation , implicating it to be representative of Ewing’s sarcoma patients. Survival was then analyzed comparing patients with positive and negative staining for cyclin D1 (Fig. 2C)Citation and VEGF (Fig. 2D)Citation . Whereas cyclin D1 expression did not significantly correlate with survival (P = 0.84), patients who had a positive staining for VEGF at the time of diagnosis had a significantly worse prognosis than patients with negative VEGF staining (P = 0.0047). Age (P = 0.28), gender (P = 0.15), anatomical location of tumors (P = 0.35), and treatment with or without chemotherapy (P = 0.67) did not significantly affect survival. Also, the combination of each of these parameters with the expression status of cyclin D1 and VEGF did not identify a survival advantage. Multivariate analysis including VEGF expression and disease status at presentation revealed that VEGF expression (P = 0.007) is a significantly better predictor of survival than disease status (P = 0.076) at the time of presentation.



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  		Fig. 2. Survival analyses. A, Kaplan-Meier survival curve of the 31 patients with Ewing’s sarcoma included in the present study. B, Kaplan-Meier curve comparing the survival of the 18 patients who presented with localized disease with that of the 13 patients presenting with metastatic disease. C, Kaplan-Meier survival curve showing patients with positive and negative staining for cyclin D1. D, Kaplan-Meier survival curve showing patients with positive and negative staining for vascular endothelial growth factor.

 
EWS-ETS Oncoproteins Activate the Cyclin D1 Promoter.
A molecular signature of Ewing’s sarcoma is the presence of EWS-ETS fusion proteins that are the result of a fusion between the Ewing’s sarcoma gene (EWS) and one of five genes encoding for ETS transcription factors (21) . These EWS-ETS oncoproteins share the same biological properties and act as aberrant transcription factors that all induce cell transformation and tumors in immunodeficient mice (30 , 31) . In this study, we focused on two of the EWS-ETS fusion proteins, the one most frequently observed in Ewing’s sarcoma patients (EWS-Fli1) and one of the rarer fusion proteins, namely, EWS-ER81 (also called EWS-ETV1). To find a molecular mechanism responsible for cyclin D1 and VEGF overexpression in Ewing’s sarcoma, we focused on the regulation of the cyclin D1 (CCND1) and VEGF genes by EWS-Fli1 and EWS-ER81.

Initial analysis indicated that the EWS-ER81 protein activated a CCND1 promoter luciferase construct (-1086/+135) by ~20-fold, whereas the parental luciferase reporter plasmid, pGL2-Basic, was negligibly affected (Fig. 3A)Citation . A comparable activation of the CCND1 promoter was observed on overexpression of EWS-Fli1 (Fig. 3B)Citation , implying that EWS-ETS oncoproteins act in the same manner to induce CCND1 gene transcription. We further analyzed the activation potential of the two portions of the EWS-ER81 fusion protein, namely, the NH2-terminal domain (NTD) encompassing EWS amino acids and the COOH-terminal domain (CTD) encompassing the ER81-derived amino acids. Neither the ER81-CTD nor the EWS-NTD portions were able to greatly enhance CCND1 promoter activity (Fig. 3B)Citation , indicating that both portions of the EWS-ER81 fusion protein are required for efficient CCND1 gene activation.



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  		Fig. 3. EWS-ETS fusion proteins activate the CCND1 promoter. A, 10 ng of EWS-ER81 expression plasmid or empty vector were transfected into RK13 cells. Activation of the cotransfected CCND1–1086 luciferase reporter plasmid or the parental luciferase reporter pGL2-Basic is shown. B, analogous activation of the CCND1–1086 promoter by EWS-Fli1, ER81-CTD, EWS-NTD, and EWS-ER81. C, schematic overview of the CCND1 promoter truncations that were used. D, activation of the indicated CCND1 promoter constructs by EWS-ER81 in RK13 cells. E, activation of the indicated CCND1 promoter constructs by EWS-Fli1 in RK13 cells.

 
To delineate elements in the CCND1 promoter that mediate stimulation by EWS-ETS proteins, we constructed several CCND1 promoter truncations (Fig. 3C)Citation . Deletion of promoter sequences from -1086 to -159 did not affect the ability of EWS-ER81 (Fig. 3D)Citation and EWS-Fli1 (Fig. 3E)Citation to activate the CCND1 promoter. In contrast, further truncating the promoter to -62 or -45 resulted in a significant decrease in promoter activity in the absence and presence of EWS-ETS fusion proteins. These results suggest that sequences between -159 and -62 in the CCND1 promoter can mediate the response to EWS-ETS oncoproteins.

Interestingly, the CCND1–159 promoter truncation does not contain any ETS consensus sequence to which EWS-ETS proteins normally bind (21) . However, it was reported that EWS-ETS fusion proteins are also capable of activating promoters independent of binding to the DNA (32) . Furthermore, two Sp1 binding sites have been identified within the region -159 to -62 that regulate CCND1 promoter activity (33, 34, 35, 36) . We hypothesized that EWS-ETS oncoproteins may mediate their effect, at least in part, through these Sp1 binding sites. Therefore, we deleted the region encompassing these two Sp1 binding sites (nucleotides -138 to -98; see {Delta}Sp1 in Fig. 4ACitation ) in the CCND1–1086 promoter construct and observed that it was only stimulated half as much by EWS-ER81 (Fig. 4B)Citation and EWS-Fli1 (Fig. 4C)Citation when compared with the wild-type promoter. Similarly, induction of the CCND1–1086{Delta}Sp1 construct by Sp1 was reduced by more than half. Furthermore, we coexpressed Sp1 with EWS-ER81 or EWS-Fli1 and found that these transcription factors collaborated in CCND1 promoter stimulation (Fig. 4, B and C)Citation . Taken together, our results indicate that EWS-ETS fusion proteins and Sp1 can jointly activate CCND1 gene transcription and that this activation involves the Sp1 binding sites within the region -138 to -98 of the CCND1 promoter.



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  		Fig. 4. EWS-ETS collaborates with Sp1 on the CCND1 promoter. A, sketch of the CCND1–1086 promoter construct or one where a region encompassing two Sp1 binding sites was deleted (nucleotides -138 to -98; {Delta}Sp1). B, EWS-ER81 and/or Sp1 was cotransfected with the indicated luciferase reporter constructs into RK13 cells. C, EWS-Fli1 and/or Sp1 was cotransfected with the indicated luciferase reporter constructs into RK13 cells.

 
Activation of the VEGF Promoter by EWS-ETS Oncoproteins.
Next, we assessed the ability of EWS-ETS oncoproteins to regulate the VEGF promoter. Indeed, EWS-ER81 activated a VEGF (-2274 to +379) promoter luciferase construct by ~6-fold (Fig. 5B)Citation ; similar data were obtained for EWS-Fli1 (see Fig. 5CCitation ). To define the elements in the VEGF promoter that are responsible for its inducibility by EWS-ER81, we constructed a series of promoter truncations (Fig. 5A)Citation . Transfection analysis with the -953 to +379 promoter construct indicated that the binding site (see HRE in Fig. 5ACitation ) for hypoxia-inducible factor, a key regulator of VEGF promoter regulation and angiogenesis (17) , has no influence in VEGF promoter activation by EWS-ETS fusion proteins (see VEGF-953 in Fig. 5, B and CCitation ). Even further truncation of the VEGF promoter down to -171 (see VEGF-449 in Fig. 5BCitation and VEGF-171 in Fig. 5CCitation ) did not affect the ability of EWS-ER81 or EWS-Fli1 to stimulate transcription. However, truncation down to -27 abolished both basal and EWS-ER81- or EWS-Fli1-stimulated VEGF promoter activity (see VEGF-27 in Fig. 5, B and CCitation ), suggesting that sequences downstream of -171 are important for EWS-ETS oncoproteins to stimulate VEGF transcription.



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  		Fig. 5. EWS-ETS fusion proteins activate the vascular endothelial growth factor (VEGF) promoter. A, schematic overview of VEGF promoter constructs used. EWS-ER81 (B) and EWS-Fli1 (C) were transfected into RK13 cells. Luciferase activities derived from the indicated cotransfected VEGF promoter constructs are shown.

 
To further delineate the promoter region that is inducible by EWS-ER81 and EWS-Fli1, we deleted sequences from +101 to +379 (sequences with the suffix "s"; see Fig. 5ACitation ) and observed that these sequences have no impact on EWS-ER81 or EWS-Fli1 inducibility of the VEGF promoter (compare VEGF-953 with VEGF-953s or VEGF-171 with VEGF-171s; Fig. 5, B and CCitation ). Thus, we surmised that promoter sequences between -171 and +100 are sufficient for EWS-ETS fusion proteins to activate the VEGF gene. Only one potential ETS binding site to which EWS-ETS oncoproteins normally bind is localized within this region and overlaps with the transcription start site. However, mutation of the respective ETS core binding site from GGAT to AAAT (nucleotides -4 to -1; prefix "m"; see Fig. 5ACitation ) did not affect the ability of EWS-ER81 to induce the VEGF promoter (compare VEGF-m953s with VEGF-953s or VEGF-m171s with VEGF-171s; Fig. 5BCitation ). Thus, we conclude that EWS-ETS oncoproteins most likely do not require direct DNA binding to the VEGF promoter to stimulate it.

Interestingly, and in accordance with the findings in the CCND1 promoter, our smallest VEGF promoter construct that is inducible by both EWS-Fli1 and EWS-ER81 (-171 to +100; VEGF-171s) encompasses a region that is highly GC rich (-109 to -37) and contains binding sites for the transcription factor Sp1. Indeed, Sp1 activation of the VEGF promoter through these sites, either alone or in conjunction with other transcription factors, has been demonstrated previously (37, 38, 39, 40) . Thus, we created a construct (VEGF-89s; see Fig. 5ACitation ) in which the upstream half of the GC-rich promoter region has been deleted. This construct was less inducible by EWS-Fli1 and EWS-ER81 than VEGF-171s, but it needed complete deletion of the GC-rich region to eliminate inducibility by these EWS-ETS proteins (see VEGF-89s and VEGF-27s in Fig. 5, B and CCitation ).

Next, we deleted a region in the VEGF promoter (-120 to -38) encompassing the GC-rich region ({Delta}Sp1 mutants). Indeed, the VEGF-171{Delta}Sp1 construct was no longer inducible by EWS-ER81 (Fig. 6A)Citation . However, the VEGF-953{Delta}Sp1 construct was as inducible by EWS-ER81 as the VEGF-953 construct (Fig. 6A)Citation . This suggests that in the longer promoter construct, other Sp1 elements can substitute for the deleted ones and help EWS-ETS to induce the VEGF promoter. Consistent with this notion, the VEGF-953{Delta}Sp1 promoter construct was still inducible by Sp1 (see Fig. 6, C or DCitation ).



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  		Fig. 6. EWS-ETS-induced activation of the vascular endothelial growth factor (VEGF) promoter is mediated mainly through Sp1 binding sites. A, 10 ng of EWS-ER81 expression plasmid or empty vector were transfected with the indicated VEGF promoter constructs into RK13 cells. B, activation of the VEGF promoter (-2274 to +379) by 50 ng of EWS-ER81 or EWS-Fli1 and 250 ng of Sp1 in RK13 cells. C, 10 ng of EWS-ER81 and 50 ng of Sp1 expression vectors were cotransfected with different VEGF promoter constructs as indicated into RK13 cells. D, 10 ng of EWS-Fli1 and 50 ng of Sp1 expression vectors were cotransfected with different VEGF promoter constructs as indicated into RK13 cells. E, a GAL4 DNA binding site-driven luciferase construct was cotransfected with 50 ng of GAL4-EWS-ER81, VP16, or VP16-Sp1 plasmids, as indicated, into Ovcar3 cells. Resulting luciferase activities are depicted.

 
We also analyzed how overexpression of Sp1 affected the ability of EWS-ETS fusion proteins to induce the VEGF promoter. As shown in Fig. 6BCitation , EWS-ER81 and Sp1, as well as EWS-Fli1 and Sp1, synergized to induce VEGF-953 transcription. Furthermore, this synergy between EWS-ER81 and EWS-Fli1 and Sp1 was also observed with the smaller promoter constructs VEGF-171 and VEGF-171s (Fig. 6, C and D)Citation . As expected, the VEGF-171{Delta}Sp1 promoter construct was not resurrected in its activity by joint overexpression of EWS-ER81 and Sp1. However, to our surprise, although VEGF-953{Delta}Sp1 was still inducible by EWS-ER81 or Sp1 alone, no synergy between these factors was observable any longer. Taken together, our results indicate that EWS-ETS fusion proteins and Sp1 synergistically activate the VEGF promoter and that Sp1 binding sites within the region -120 to -38 appear to be important for this synergy.

Interaction of EWS-ER81 with Sp1.
We wondered how EWS-ETS fusion proteins and Sp1 may synergize and asked whether a direct protein-protein interaction is involved. To test this hypothesis, we performed a mammalian two-hybrid assay. To this end, EWS-ER81 was fused to the DNA-binding domain of the yeast transcription factor GAL4. This GAL4-EWS-ER81 fusion protein enhanced transcription of a luciferase reporter gene driven by GAL4 DNA binding sites (Fig. 6E)Citation . We then coexpressed the potent transactivation domain of VP16 with GAL4-EWS-ER81, but no significant changes in luciferase activity were observed. However, when VP16 was fused to Sp1, it stimulated GAL4-EWS-ER81-dependent transcription by 5.5-fold (Fig. 6E)Citation , whereas VP16-Sp1 had little effect on the GAL4 moiety, indicating that Sp1 binds to EWS-ER81. Thus, the Ewing’s sarcoma-associated EWS-ETS fusion proteins may not only synergize but also physically interact with Sp1.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we analyzed Ewing’s sarcoma specimens for the overexpression of four proteins that can play critical roles in carcinogenesis but are only poorly characterized in Ewing’s sarcoma. The first protein we studied was HER2/Neu. Intriguingly, HER2/Neu overexpression was found previously in Ewing’s sarcoma cell lines, which is in contrast to other reports analyzing HER2/Neu expression in patient samples (41, 42, 43, 44) . Our analyses support the notion that HER2/Neu is not overexpressed in Ewing’s sarcomas and points out the limitations of basing conclusions merely on cell lines. Similarly, Mdm2 expression in Ewing’s sarcoma has only been sparsely analyzed, with contradictory results. Whereas three studies (45, 46, 47) found no overexpression of Mdm2, both Radig et al. (48) and Ladanyi et al. (49) did, and the latter study reported it in 10% of all analyzed samples (n = 30). In our series, none of the 31 Ewing’s sarcoma specimens displayed Mdm2 overexpression. This finding supports the idea that Mdm2 plays a minor role in Ewing’s sarcoma carcinogenesis and that inactivation of p53 in Ewing’s sarcoma may be caused by mechanisms other than Mdm2 overexpression.

To our knowledge, cyclin D1 protein expression has not been analyzed in Ewing’s sarcoma, although microarray analyses showed an overexpression of CCND1 in Ewing’s sarcoma (6 , 7) , and high levels of CCND1 mRNA expression in patient samples were reported (50) , which appears to be independent of any CCND1 gene amplification (47) . Consistently, we found that cyclin D1 protein was overexpressed in 42% of our patients, implying that cyclin D1 overexpression is of importance in Ewing’s sarcoma. However, we were unable to correlate cyclin D1 expression with any clinical parameter, implying that cyclin D1 expression is not a useful tumor marker.

Finally, we analyzed the overexpression of VEGF in Ewing’s sarcoma tumor samples. We found VEGF protein to be overexpressed in 55% of Ewing’s sarcoma specimens, thereby validating previous data derived from the transcriptional profiling of an EWS-Fli1-overexpressing cell line (6) . Importantly, survival analyses revealed that VEGF overexpression is strongly correlated with poor survival. It might be hypothesized that this finding may be different in a patient group treated today because of different treatment modalities. However, we found no difference in survival between those patients who had received chemotherapy and those who had not received chemotherapy. Furthermore, the 5-year survival rate of Ewing’s sarcoma patients has not changed dramatically over the last two decades (27) . We conclude that if the correlation between VEGF overexpression and poor survival is substantiated in a larger patient pool from several centers, VEGF may become a reliable predictor of survival and aid in the treatment of Ewing’s sarcoma patients.

VEGF overexpression has been shown to be correlated to poor prognosis in a variety of different cancers. Interestingly, elevated levels of VEGF in the serum have also proven their utility as a marker of tumor progression (51) . Of note, elevated serum levels of VEGF in patients with Ewing’s sarcoma were reported previously (52, 53, 54) . However, the variability in VEGF serum levels of Ewing’s sarcoma patients as well as healthy children was very high, and the low number of cases studied precludes a sound judgment as to whether VEGF serum levels could serve as a tumor marker for Ewing’s sarcoma. However, VEGF serum levels should be studied in a larger cohort of Ewing’s sarcoma patients and correlated with survival data because the noninvasive determination of VEGF overexpression by measuring serum levels would be more convenient than immunohistochemical staining of tumor specimens.

By promoter analyses, we tried to find an explanation for the overexpression of cyclin D1 and VEGF in Ewing’s sarcoma and found a common mechanism. Both the CCND1 and VEGF promoters were activated indiscriminately by EWS-Fli1 and EWS-ER81, further substantiating the notion that all five known EWS-ETS fusion proteins act in the same manner to induce Ewing’s sarcoma. However, because only approximately half of all Ewing’s sarcomas displayed overexpression of cyclin D1 and VEGF, EWS-ETS proteins per se are not sufficient to stimulate CCND1 and VEGF expression. It appears that the cellular environment is different in different Ewing’s sarcoma tissues, being either conducive or prohibitive for the induction of CCND1 and VEGF transcription, which might be reflective of different tissues of origin of the Ewing’s sarcomas studied.

Interestingly, our findings imply that EWS-ETS fusion proteins activate the CCND1 and VEGF promoters without direct DNA binding because even promoter constructs without any potential ETS binding sites were stimulated by EWS-Fli1 and EWS-ER81. This is in contrast to the finding of Fukuma et al. (55) with respect to the CCND1 promoter; they showed binding of EWS-Fli1 to the -1087 to -742 fragment of the CCND1 promoter by chromatin immunoprecipitation assay. However, our results indicated that this region of the CCND1 promoter is not required for transcriptional activation of the CCND1 promoter, but promoter sequences between -159 and -62 that encompass a GC-rich region are required. Similarly, VEGF promoter stimulation by EWS-Fli1 and EWS-ER81 depended on a GC-rich region between -171 and -27. Both of these GC-rich regions contain binding sites for the transcription factor Sp1, which has been shown to play a critical role in the transcriptional regulation of the CCND1 and VEGF promoters (33, 34, 35 , 37 , 39 , 40 , 56 , 57) . Consistently, we found that Sp1 activated both promoters, and importantly, Sp1 synergized with EWS-ETS fusion proteins to do so. Potentially, because Sp1 and EWS-ER81 interacted in a mammalian two-hybrid assay, Sp1 may recruit EWS-ETS fusion proteins to a gene promoter by direct protein-protein interactions, thereby providing one possible explanation why EWS-ETS fusion proteins do not require direct DNA binding for VEGF and CCND1 promoter activation. Because Sp1 plays important roles in carcinogenesis (58 , 59) , additional studies of how Sp1 and EWS-ETS fusion proteins synergize and whether this synergy may be observed at a wide range of gene promoters will enhance our understanding of Ewing’s sarcoma development.


   ACKNOWLEDGMENTS
 
We thank Drs. Spelsberg and Semenza for kindly providing reagents.


   FOOTNOTES
 
Grant support: Stipend (to B. F.) from the Swiss Orthopedic Society and Grant CA085257 (to R. J.) from the National Cancer Institute.

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.

Requests for reprints: Ralf Janknecht, Department of Biochemistry and Molecular Biology, Mayo Clinic, Guggenheim 1501A Building, 200 First Street SW, Rochester, Minnesota 55905. Phone: (507) 266-4393; Fax: (507) 284-1767; E-mail: janknecht.ralf{at}mayo.edu

Received 8/13/03; revised 11/ 3/03; accepted 11/11/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Delattre O., Zucman J., Plougastel B., Desmaze C., Melot T., Peter M., Kovar H., Joubert I., de Jong P., Rouleau G., et al Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature (Lond.), 359: 162-165, 1992.[CrossRef][Medline]
  2. Sidransky D. Emerging molecular markers of cancer. Nat. Rev. Cancer, 2: 210-219, 2002.[CrossRef][Medline]
  3. Pollack J. R., Perou C. M., Alizadeh A. A., Eisen M. B., Pergamenschikov A., Williams C. F., Jeffrey S. S., Botstein D., Brown P. O. Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat. Genet., 23: 41-46, 1999.[Medline]
  4. Schena M. Genome analysis with gene expression microarrays. Bioessays, 18: 427-431, 1996.[CrossRef][Medline]
  5. Wai D. H., Schaefer K. L., Schramm A., Korsching E., Van Valen F., Ozaki T., Boecker W., Schweigerer L., Dockhorn-Dworniczak B., Poremba C. Expression analysis of pediatric solid tumor cell lines using oligonucleotide microarrays. Int. J. Oncol., 20: 441-451, 2002.[Medline]
  6. Lessnick S. L., Dacwag C. S., Golub T. R. The Ewing’s sarcoma oncoprotein EWS/FLI induces a p53-dependent growth arrest in primary human fibroblasts. Cancer Cell, 1: 393-401, 2002.[CrossRef][Medline]
  7. Khan J., Wei J. S., Ringner M., Saal L. H., Ladanyi M., Westermann F., Berthold F., Schwab M., Antonescu C. R., Peterson C., Meltzer P. S. Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks. Nat. Med., 7: 673-679, 2001.[CrossRef][Medline]
  8. Sherr C. J., McCormick F. The RB and p53 pathways in cancer. Cancer Cell, 2: 103-112, 2002.[CrossRef][Medline]
  9. Yarden Y., Sliwkowski M. X. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell. Biol., 2: 127-137, 2001.[CrossRef][Medline]
  10. Yarden Y. The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur. J. Cancer, 37 (Suppl. 4): S3-S8, 2001.
  11. Blagosklonny M. V., Pardee A. B. The restriction point of the cell cycle. Cell Cycle, 1: 103-110, 2002.[Medline]
  12. Holash J., Wiegand S. J., Yancopoulos G. D. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene, 18: 5356-5362, 1999.[CrossRef][Medline]
  13. Ferrara N., Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nat. Med., 5: 1359-1364, 1999.[CrossRef][Medline]
  14. D’Amore P. A., Ng Y. S. Won’t you be my neighbor? Local induction of arteriogenesis. Cell, 110: 289-292, 2002.[CrossRef][Medline]
  15. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am. J. Physiol. Cell Physiol., 280: C1358-C1366, 2001.[Abstract/Free Full Text]
  16. Carmeliet P., Jain R. K. Angiogenesis in cancer and other diseases. Nature (Lond.), 407: 249-257, 2000.[CrossRef][Medline]
  17. Harris A. L. Hypoxia: a key regulatory factor in tumour growth. Nat. Rev. Cancer, 2: 38-47, 2002.[CrossRef][Medline]
  18. Ruoslahti E. Specialization of tumour vasculature. Nature Rev. Cancer, 2: 83-90, 2002.[CrossRef][Medline]
  19. Bruick R. K., McKnight S. L. Building better vasculature. Genes Dev., 15: 2497-2502, 2001.[Free Full Text]
  20. Ladanyi M. EWS-FLI1 and Ewing’s sarcoma: recent molecular data and new insights. Cancer Biol. Ther., 1: 330-336, 2002.[Medline]
  21. Arvand A., Denny C. T. Biology of EWS/ETS fusions in Ewing’s family tumors. Oncogene, 20: 5747-5754, 2001.[CrossRef][Medline]
  22. Kumar R., Schaefer J., Grande J. P., Roche P. C. Immunolocalization of calcitriol receptor, 24-hydroxylase cytochrome P- 450, and calbindin D28k in human kidney. Am. J. Physiol., 266: F477-F485, 1994.
  23. Forsythe J. A., Jiang B. H., Iyer N. V., Agani F., Leung S. W., Koos R. D., Semenza G. L. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol., 16: 4604-4613, 1996.[Abstract]
  24. Bredemeier-Ernst I., Nordheim A., Janknecht R. Transcriptional activity and constitutive nuclear localization of the ETS protein Elf-1. FEBS Lett., 408: 47-51, 1997.[CrossRef][Medline]
  25. Kovar H., Dworzak M., Strehl S., Schnell E., Ambros I. M., Ambros P. F., Gadner H. Overexpression of the pseudoautosomal gene MIC2 in Ewing’s sarcoma and peripheral primitive neuroectodermal tumor. Oncogene, 5: 1067-1070, 1990.[Medline]
  26. Perlman E. J., Dickman P. S., Askin F. B., Grier H. E., Miser J. S., Link M. P. Ewing’s sarcoma: routine diagnostic utilization of MIC2 analysis: a Pediatric Oncology Group/Children’s Cancer Group Intergroup Study. Hum. Pathol., 25: 304-307, 1994.[CrossRef][Medline]
  27. Grier H. E., Krailo M. D., Tarbell N. J., Link M. P., Fryer C. J., Pritchard D. J., Gebhardt M. C., Dickman P. S., Perlman E. J., Meyers P. A., Donaldson S. S., Moore S., Rausen A. R., Vietti T. J., Miser J. S. Addition of ifosfamide and etoposide to standard chemotherapy for Ewing’s sarcoma and primitive neuroectodermal tumor of bone. N. Engl. J. Med., 348: 694-701, 2003.[Abstract/Free Full Text]
  28. Paulussen M., Ahrens S., Dunst J., Winkelmann W., Exner G. U., Kotz R., Amann G., Dockhorn-Dworniczak B., Harms D., Muller-Weihrich S., Welte K., Kornhuber B., Janka-Schaub G., Gobel U., Treuner J., Voute P. A., Zoubek A., Gadner H., Jurgens H. Localized Ewing tumor of bone: final results of the cooperative Ewing’s Sarcoma Study CESS 86. J. Clin. Oncol., 19: 1818-1829, 2001.[Abstract/Free Full Text]
  29. Riley R. D., Burchill S. A., Abrams K. R., Heney D., Sutton A. J., Jones D. R., Lambert P. C., Young B., Wailoo A. J., Lewis I. J. A systematic review of molecular and biological markers in tumours of the Ewing’s sarcoma family. Eur. J. Cancer, 39: 19-30, 2003.
  30. Thompson A. D., Teitell M. A., Arvand A., Denny C. T. Divergent Ewing’s sarcoma EWS/ETS fusions confer a common tumorigenic phenotype on NIH3T3 cells. Oncogene, 18: 5506-5513, 1999.[CrossRef][Medline]
  31. May W. A., Lessnick S. L., Braun B. S., Klemsz M., Lewis B. C., Lunsford L. B., Hromas R., Denny C. T. 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., 13: 7393-7398, 1993.[Abstract/Free Full Text]
  32. Welford S. M., Hebert S. P., Deneen B., Arvand A., Denny C. T. DNA binding domain-independent pathways are involved in EWS/FLI1-mediated oncogenesis. J. Biol. Chem., 276: 41977-41984, 2001.[Abstract/Free Full Text]
  33. Yan G. Z., Ziff E. B. Nerve growth factor induces transcription of the p21 WAF1/CIP1 and cyclin D1 genes in PC12 cells by activating the Sp1 transcription factor. J. Neurosci., 17: 6122-6132, 1997.[Abstract/Free Full Text]
  34. Nagata D., Suzuki E., Nishimatsu H., Satonaka H., Goto A., Omata M., Hirata Y. Transcriptional activation of the cyclin D1 gene is mediated by multiple cis-elements, including SP1 sites and a cAMP-responsive element in vascular endothelial cells. J. Biol. Chem., 276: 662-669, 2001.[Abstract/Free Full Text]
  35. Lin H. M., Pestell R. G., Raz A., Kim H. R. Galectin-3 enhances cyclin D1 promoter activity through SP1 and a cAMP-responsive element in human breast epithelial cells. Oncogene, 21: 8001-8010, 2002.[CrossRef][Medline]
  36. Hilton T. L., Wang E. H. Transcription factor IID recruitment and Sp1 activation. Dual function of TAF1 in cyclin D1 transcription. J. Biol. Chem., 278: 12992-13002, 2003.[Abstract/Free Full Text]
  37. Shi Q., Le X., Abbruzzese J. L., Peng Z., Qian C. N., Tang H., Xiong Q., Wang B., Li X. C., Xie K. Constitutive Sp1 activity is essential for differential constitutive expression of vascular endothelial growth factor in human pancreatic adenocarcinoma. Cancer Res., 61: 4143-4154, 2001.[Abstract/Free Full Text]
  38. Schafer G., Cramer T., Suske G., Kemmner W., Wiedenmann B., Hocker M. Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements. J. Biol. Chem., 278: 8190-8198, 2003.[Abstract/Free Full Text]
  39. Finkenzeller G., Sparacio A., Technau A., Marme D., Siemeister G. Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression. Oncogene, 15: 669-676, 1997.[CrossRef][Medline]
  40. Mukhopadhyay D., Knebelmann B., Cohen H. T., Ananth S., Sukhatme V. P. The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity. Mol. Cell. Biol., 17: 5629-5639, 1997.[Abstract]
  41. Thomas D. G., Giordano T. J., Sanders D., Biermann J. S., Baker L. Absence of HER2/neu gene expression in osteosarcoma and skeletal Ewing’s sarcoma. Clin. Cancer Res., 8: 788-793, 2002.[Abstract/Free Full Text]
  42. Amir G., Issakov J., Meller I., Sucher E., Peyser A., Cohen I. J., Yaniv I., Ben Arush M. W., Tavori U., Kollender Y., Ron N., Peylan-Ramu N. Expression of p53 gene product and cell proliferation marker Ki-67 in Ewing’s sarcoma: correlation with clinical outcome. Hum. Pathol., 33: 170-174, 2002.[CrossRef][Medline]
  43. Zhou R. R., Jia S. F., Zhou Z., Wang Y., Bucana C. D., Kleinerman E. S. Adenovirus-E1A gene therapy enhances the in vivo sensitivity of Ewing’s sarcoma to VP-16. Cancer Gene Ther., 9: 407-413, 2002.[CrossRef][Medline]
  44. Zhou Z., Jia S. F., Hung M. C., Kleinerman E. S. E1A sensitizes HER2/neu-overexpressing Ewing’s sarcoma cells to topoisomerase II-targeting anticancer drugs. Cancer Res., 61: 3394-3398, 2001.[Abstract/Free Full Text]
  45. Tsuchiya T., Sekine K., Hinohara S., Namiki T., Nobori T., Kaneko Y. Analysis of the p16INK4, p14ARF, p15, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma. Cancer Genet. Cytogenet., 120: 91-98, 2000.[CrossRef][Medline]
  46. Kovar H., Auinger A., Jug G., Aryee D., Zoubek A., Salzer-Kuntschik M., Gadner H. Narrow spectrum of infrequent p53 mutations and absence of MDM2 amplification in Ewing tumours. Oncogene, 8: 2683-2690, 1993.[Medline]
  47. Lopez-Guerrero J. A., Pellin A., Noguera R., Carda C., Llombart-Bosch A. Molecular analysis of the 9p21 locus and p53 genes in Ewing family tumors. Lab. Investig., 81: 803-814, 2001.[Medline]
  48. Radig K., Schneider-Stock R., Rose I., Mittler U., Oda Y., Roessner A. p53 and ras mutations in Ewing’s sarcoma. Pathol. Res. Pract., 194: 157-162, 1998.[Medline]
  49. Ladanyi M., Lewis R., Jhanwar S. C., Gerald W., Huvos A. G., Healey J. H. MDM2 and CDK4 gene amplification in Ewing’s sarcoma. J. Pathol., 175: 211-217, 1995.[CrossRef][Medline]
  50. Matsumoto Y., Tanaka K., Nakatani F., Matsunobu T., Matsuda S., Iwamoto Y. Downregulation and forced expression of EWS-Fli1 fusion gene results in changes in the expression of G1 regulatory genes. Br. J. Cancer, 84: 768-775, 2001.[CrossRef][Medline]
  51. Poon R. T., Fan S. T., Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J. Clin. Oncol., 19: 1207-1225, 2001.[Abstract/Free Full Text]
  52. Holzer G., Obermair A., Koschat M., Preyer O., Kotz R., Trieb K. Concentration of vascular endothelial growth factor (VEGF) in the serum of patients with malignant bone tumors. Med. Pediatr. Oncol., 36: 601-604, 2001.[CrossRef][Medline]
  53. Kushlinskii N. E., Babkina I. V., Solov’ev Y. N., Trapeznikov N. N. Vascular endothelium growth factor and angiogenin in the serum of patients with osteosarcoma and Ewing’s tumor. Bull. Exp. Biol. Med., 130: 691-693, 2000.[Medline]
  54. Pavlakovic H., Von Schutz V., Rossler J., Koscielniak E., Havers W., Schweigerer L. Quantification of angiogenesis stimulators in children with solid malignancies. Int. J. Cancer, 92: 756-760, 2001.[CrossRef][Medline]
  55. Fukuma M., Okita H., Hata J., Umezawa A. Upregulation of Id2, an oncogenic helix-loop-helix protein, is mediated by the chimeric EWS/ets protein in Ewing sarcoma. Oncogene, 22: 1-9, 2003.[CrossRef][Medline]
  56. Ryuto M., Ono M., Izumi H., Yoshida S., Weich H. A., Kohno K., Kuwano M. Induction of vascular endothelial growth factor by tumor necrosis factor {alpha} in human glioma cells. Possible roles of SP-1. J. Biol. Chem., 271: 28220-28228, 1996.[Abstract/Free Full Text]
  57. Silins G., Grimmond S., Egerton M., Hayward N. Analysis of the promoter region of the human VEGF-related factor gene. Biochem. Biophys. Res. Commun., 230: 413-418, 1997.[CrossRef][Medline]
  58. Kaczynski J., Cook T., Urrutia R. Sp1- and Kruppel-like transcription factors. Genome Biol., 4: 206 2003.[CrossRef][Medline]
  59. Black A. R., Black J. D., Azizkhan-Clifford J. Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer. J. Cell. Physiol., 188: 143-160, 2001.[CrossRef][Medline]



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