Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, Texas
Corresponding author: Eugenie S. Kleinerman, MD, Division of Pediatrics, Unit 87, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; Fax: (713) 794-5042; email@example.com
We thank Dr. Vidya Gopalakrishnan for helpful suggestions and Ms. Jeanette Quimby for article preparation.
RE1-silencing transcription factor (REST), a neuronal repressor gene, regulates neuronal stem cell differentiation. Ewing sarcoma may originate from neural crest cells. In the current study, the authors investigated whether REST plays a role in the growth of this tumor.
REST expression was determined by Western blot analysis and reverse transcription-polymerase chain reaction in 3 human Ewing sarcoma cell lines and 7 patient tumor samples. The role of REST in tumor growth and tumor vascular morphology was determined using a Ewing sarcoma xenograft model. Immunofluorescence staining, Hypoxyprobe, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed to investigate the impact of REST on pericyte marker expression, hypoxia, and apoptosis in vivo.
High levels of REST were expressed in all 3 human Ewing sarcoma cell lines and in 6 of the 7 patient tumor samples. Overexpression of EWS-FLI-1 in human mesenchymal stem cells and human neural progenitor cells was found to increase REST expression. Inhibition of EWS-FLI-1 using small interfering RNA decreased REST expression in human Ewing sarcoma cells. Inhibition of REST did not affect EWS-FLI-1, but significantly suppressed tumor growth in vivo, reduced the tumor vessel pericyte markers α- smooth muscle actin (SMA) and desmin, increased hypoxia and apoptosis in tumor tissues, and decreased the expression of delta-like ligand 4 (DLL4) and Hes1.
Ewing sarcoma is the second most common malignant bone tumor in children and young adults, with the lung being the most common site of metastasis. The survival rates for patients with metastatic disease have not improved in over 20 years.[1-3] Understanding the biology of this tumor is critical to the identification of new therapeutic targets. The origin of Ewing sarcoma is still controversial. However, several data have supported its origin in the neural crest.[4-6] Therefore, genes that control neuronal stem cell differentiation may play a role in the tumorigenesis of Ewing sarcoma.
RE1-silencing transcription factor (REST) is a neuronal repressor gene that regulates neuronal stem cell differentiation.[7, 8] Recent studies have demonstrated that REST also plays a multifunctional role in the regulation of nonneurogenic cells.[9, 10] In tumor growth, REST has a dual function depending on the cellular context. Previous studies have indicated that REST is an oncogene for medulloblastoma, a pediatric brain tumor. REST expression is elevated in samples from patients with medulloblastoma and in tumor cell lines. Abnormal expression of REST and Myc in neural progenitor cells has been shown to induce cerebellar tumors by blocking neuronal differentiation. However, the role of REST in Ewing sarcoma, which may originate from the neural crest and share neural marker features, has not been elucidated to date.
In the current study, we demonstrated that EWS-FLI-1, the hallmark fusion protein of Ewing sarcoma, regulates REST expression. Tumor samples from patients with Ewing sarcoma and cell lines expressed high levels of REST. Inhibition of EWS-FLI-1 resulted in decreased REST expression. The inhibition of REST did not affect EWS-FLI-1 levels, but did result in suppressed tumor growth, decreased tumor vessel pericyte marker expression, and increased tumor hypoxia and apoptosis. Our previous studies indicated that the Notch pathway, specifically delta-like ligand 4 (DLL4) and Hes1, is critical in the regulation of vessel pericyte formation. Herein, we further showed that inhibition of REST reduced DLL4 and Hes1 expression. These results suggested that REST is a potential new therapeutic target for the treatment of Ewing sarcoma.
MATERIALS AND METHODS
TC71 and A4573 human Ewing sarcoma cell lines were authenticated by short terminal repeat fingerprinting in the core facility. SK-ES human Ewing sarcoma cells, human mesenchymal stem cells (hMSC), human normal osteoblasts, Daoy human medulloblastoma cells, and SAOS-2 human osteosarcoma cells were purchased from the American Type Culture Collection (Manassas, Va). Human neural progenitor cells (HNP) were purchased from Lonza Walkerville, Inc. (Walkerville, Md). The cells were maintained in the medium according to the company's instructions. All of the cells were Mycoplasma free as determined with the MycoAlert Mycoplasma Detection Kit (Lonza Group Ltd, Basel, Switzerland).
Patient Samples and Reverse Transcription-Polymerase Chain Reaction
Seven tumor specimens from patients with Ewing sarcoma were obtained from The University of Texas MD Anderson Cancer Center (Houston, Tex) and Memorial Sloan-Kettering Cancer Center (New York, NY) with the approval of the Institutional Review Boards of both institutions. RNA was extracted from human tissue samples using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, Calif). cDNA was synthesized using the Reverse Transcription System (Promega, Madison, Wis). The products were amplified by polymerase chain reaction (PCR) using specific primers for REST (sense: 5′-GGATGTGGCTGGAAAGAAAA-3′; antisense: 5-GCTGTCAACTTCCAGCTTCC-3′) and EWS-FLI-1 (sense: 5′-GCCTCCTATGCAGCTCAGTC-3′; antisense: 5-GGTTGTAACCCCCTGTGCTA-3′). The 18 S ribosomal RNA (18S) primers (Ambion Inc, Austin, Tex) were used as internal controls.
Plasmids and the Small Interfering REST Stable Transfected Cell Line
Plasmid pG5–EWS-FLI-1 was constructed by subcloning EWS-FLI-1 cDNA into the pG5-FL2 backbone plasmid at the EcoRI and HindIII sites, and the fragment was verified by sequencing. hMSC or HNP cells were transfected with pG5–EWS-FLI-1 or a control plasmid; the protein or RNA was extracted from the cells 48 hours after transfection. The pSilencer 2.1-U6 hygro-based small interfering (si) RNA (siRNA) expression vector was purchased from Ambion Inc. siRNA expression vectors targeting human REST (siREST) or EWS-FLI-1 (siEWS-FLI-1) were constructed according to the manufacturer's instructions. All inserts were verified by DNA sequencing. TC71 cells were transfected with siREST or scrambled siRNA as siControl, various clones were selected in hygromycin B, and several stable transfected clones were tested for REST expression by Western blot analysis. Stable transfected cells were cultured in medium with hygromycin B at 400 μg /mL.
Western Blot Analysis
Cells were cultured in 100-mm dishes. Cell lysate was collected in 48 hours after transfection The protein (100 μg) was loaded onto an 10% sodium dodecyl sulfate-polyacrylamide gel. Specific protein bands were detected with antihuman REST (Millipore, Temecula, Calif) and β-actin (Sigma-Aldrich, St. Louis, Mo) antibodies. Densitometric analysis was performed, and the values were normalized with β-actin loading control.
Athymic (T-cell deficient) nude mice aged 4 to 5 weeks were purchased from the National Cancer Institute (Bethesda, Md). The mice were maintained in a specific pathogen-free animal facility approved by the American Association for Assessment and Accreditation of Laboratory Animal Care. The animal experiment protocol was approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. TC71-siControl and TC71-siREST Ewing sarcoma cells in the middle of the logarithmic growth phase were harvested by trypsinization. Cell suspensions (2 × 106 cells in 0.1 mL of Hanks solution) were injected subcutaneously into the nude mice. Tumors were measured twice a week with a caliper, and diameters were recorded. Tumor volume was calculated by the formula a2b/2, in which a and b are the 2 largest diameters. The tumor tissue was collected for immunohistochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay.
Frozen tumor sections were fixed with acetone and chloroform. The sections were incubated with rat antimouse CD31 antibody (BD Biosciences, San Diego, Calif), desmin, or α-smooth muscle actin (α-SMA) antibody (Abcam, Inc, Cambridge, Mass), or DLL4 or vascular endothelial growth factor (VEGF) antibody (Santa Cruz Biotechnology, Santa Cruz, Calif). Anticyanine 5 (anti-Cy5) was used as the secondary antibody. For double fluorescence staining, the sections were first incubated with CD31 and Cy5 and then with α-SMA and Cy3 antibodies. All sections were analyzed by confocal microscopy (Carl Zeiss MicroImaging, Inc, Thornwood, NY). Relative expression was quantified in at least 5 different microscopy fields from different samples using Simple PCI software (Hamamatsu, Sewickley, PA), and the average expression was calculated.
The Hypoxyprobe-1 (pimonidazole hydrochloride; HPI) kit was purchased from HPI, Inc (Burlington, Mass). Hypoxyprobe was reconstituted in phosphate-buffered saline at a final concentration of 7 mg/mL. Mice were injected with 200 μL of Hypoxyprobe solution and sacrificed within 2.5 hours. Tumor tissues were collected for immunofluorescence staining using an antipimonidazole monoclonal antibody.
Apoptotic cells were quantified by the TUNEL assay. TUNEL staining in frozen tumor tissues was performed according to the manufacturer's instructions. The green fluorescence of apoptotic cells was detected using a fluorescence microscope. The average number of apoptotic cells in control samples and TC71-siREST tumors was calculated by counting the number of TUNEL-positive cells in 5 random microscopic fields from different samples.
A 2-tailed Student t test was used to statistically evaluate all experimental results. A P value < .05 was considered to be statistically significant.
Expression of REST in Ewing Sarcoma Cell Lines and Patient Samples
Three different human Ewing sarcoma cell lines were analyzed for REST expression at both the RNA and protein levels (Fig. 1A and 1B). TC71, A4573, and SK-ES human Ewing sarcoma cells expressed both REST and EWS-FLI-1 RNA (Fig. 1A). By contrast, normal human osteoblasts and human osteosarcoma cells (SAOS-2) did not express either REST or EWS-FLI-1 (Fig. 1A). Western blot analysis (Fig. 1B) confirmed that TC71, A4573, and SK-ES cells expressed high levels of REST protein. Daoy human medulloblastoma cells, which overexpress REST, served as the positive control. To investigate whether REST was also expressed in tumors from patients with Ewing sarcoma, total RNA was extracted from 7 different patient tumor samples and analyzed by reverse transcription (RT)-PCR. Six of the 7 patient samples expressed REST (Fig. 1C). These data demonstrated that REST is expressed both in human Ewing sarcoma cell lines and in patient tumor samples but not in normal bone cells or SAOS-2 osteosarcoma cells.
EWS-FLI-1 Regulates REST Expression
The expression of the EWS-FLI-1 fusion protein is the hallmark of Ewing sarcoma. To confirm the link between EWS-FLI-1 expression and REST expression (Fig. 1A), hMSC or HNP were transfected with pG5–EWS-FLI-1 or a control vector. As shown in Figure 2A, both EWS-FLI-1 and REST expression levels were elevated in the cells after transfection with pG5–EWS-FLI-1, but not after transfection with the control vector. REST protein levels were also increased in EWS-FLI-1–transfected hMSC cells (Fig. 2B). To determine whether inhibition of EWS-FLI-1 inhibits REST expression, we transfected 2 different human Ewing sarcoma cell lines, TC71 cells and A4573, with the siEWS-FLI-1 vector or siControl vectors. EWS-FLI-1 expression was found to be downregulated in both TC71- and A4573–siEWS-FLI-1–transfected cell lines. REST expression was also found to be decreased in these cells compared with that in control transfected cells (Fig. 2C). Different transfection efficiency of siEWS-FLI-1 induced different downregulation levels of REST between the 2 cell lines. By contrast, the transfection of TC71 cells with siREST did not appear to affect EWS-FLI-1 expression (Fig. 2D). These results indicate that EWS-FLI-1 regulates REST expression and that REST may be one of the downstream genes targeted by EWS-FLI-1.
Inhibition of REST Reduced TC71 Tumor Growth In Vivo
To determine whether REST expression contributes to tumor growth, TC71 cells were transfected with siREST, and selected clones were analyzed using Western blot analysis. REST expression levels in TC71-siREST clone 5 and clone 8 cells were reduced by 70% and 60%, respectively (Fig. 3A). We used clone 5 in our in vivo experiments. Immunofluorescence staining confirmed that REST expression was inhibited in TC71-siREST clone 5 cells compared with that in TC71-siControl cells (Fig. 3B). TC71-siREST clone 5 cells were then injected into nude mice. Tumor growth and average tumor size in the animals injected with clone 5 cells were significantly decreased compared with those in animals injected with control-transfected cells (Fig. 3C). These results suggest that inhibition of REST suppressed tumor growth.
Inhibition of REST decreased pericyte marker expression in tumor tissue
Tumor growth requires oxygen, which is delivered to tumor cells by tumor vessels. Blood vessels consist of both endothelial cells and pericytes. We therefore investigated the role of REST in the formation of tumor vessel endothelial cells and pericytes. The expression of endothelial marker CD31 was not found to be changed in TC71-siREST clone 5 tumor tissues (Fig. 4A). VEGF expression in TC71-siREST tumors was also not different from that in control tumors (Fig. 4A). However, the expression levels of the pericyte markers α-SMA and desmin were both lower in TC71-siREST tumors compared with control tumors (Fig. 4B). Quantitative data confirmed that the inhibition of REST significantly reduced the expression of both α-SMA and desmin in tumor tissues (Fig. 4C). Colocalization staining demonstrated that the expression of α-SMA, but not CD31, was reduced in TC71-siREST tumors (Fig. 4D). These results indicate that inhibition of REST decreased pericyte coverage of tumor vessels but not the number of tumor vessels. The lack of pericyte support may affect the functioning of the tumor vessels and, in turn, tumor growth.
Blockade of REST Induced Tumor Hypoxia and Apoptosis
To determine whether decreased pericyte coverage correlates with increased tumor hypoxia, the tumor sections were analyzed by immunofluorescence staining using the hypoxia probe HPI. Increased HPI expression was observed in TC71-siREST tumor samples (Fig. 5A). Furthermore, quantitative data confirmed that inhibition of REST correlated with increased hypoxia in the tumor tissues (P < .01) (Fig. 5B). To investigate the correlation between pericyte marker expression and hypoxia, double staining of desmin and HPI was performed. Decreased desmin expression and increased hypoxia (Fig. 5C), as well as increased apoptosis measured by the TUNEL assay (Figs. 5D and 5E), were noted in TC71-siREST tumor samples compared with control tumor samples. These results suggest that inhibition of REST may decrease tumor growth by altering tumor vessel morphology, which leads to increased tumor hypoxia and apoptosis.
Suppression of REST Reduced DLL4 and Hes1 Expression in TC71 Tumors
Our previous studies demonstrated that the Notch pathway, specifically DLL4, plays a critical role in tumor vessel pericyte coverage. Inhibition of DLL4 resulted in decreased pericyte marker expression in Ewing tumors in vivo, changed tumor vessel morphology, and increased tumor hypoxia. Because pericyte levels were found to be significantly decreased in siREST tumors, we next investigated whether inhibition of REST affects DLL4 and Hes1, one of the downstream effectors of the DLL4-Notch signaling pathway. Both DLL4 and Hes1 expression was found to be reduced in TC71-siREST cells (Fig. 6A). Immunofluorescence staining (Fig. 6B) demonstrated that DLL4 expression was lower in TC71-siREST tumor tissues than in siControl tumors. The Ewing sarcoma cell line A4573 was also tested. The results indicated that inhibition of REST suppressed DLL4 and Hes1 expression in both TC71 and A4573 cells (Fig. 6C). To confirm the link between EWS-FLI-1 and the Notch signaling pathway, hMSC were transfected with pG5–EWS-FLI-1 or the control plasmid. EWS-FLI-1, REST, Notch1, and Hes1 expression levels were all found to be higher after transfection with pG5–EWS-FLI-1 compared with after transfection with the pG5 control (Fig. 6D). These data suggest that downregulation of REST inhibited expression of both DLL4 and Hes1, in agreement with our previous findings that pericyte marker expression and tumor vascular pericyte coverage in Ewing sarcoma are controlled by DLL4 and Hes1.15,17
Ewing sarcoma is a poorly differentiated, small round cell tumor with expression of the neural cell markers S-100, synaptophysin, neural-specific enolase, and CD57.1,18 We investigated the role of the neuronal repressor gene REST in Ewing sarcoma to improve our understanding of the biology of this tumor. To the best of our knowledge, the current study is the first to demonstrate that high levels of REST are expressed in Ewing sarcoma cell lines and patient tumor samples, and that the expression of REST is correlated with EWS-FLI-1, the hallmark fusion protein in Ewing sarcoma. We further demonstrated that EWS-FLI-1 regulates REST expression. The transfection of EWS-FLI-1 into hMSC, in which REST is not expressed, induced REST mRNA and protein production. To determine whether this regulation is also functional in neural cells, these results were confirmed using HNP cells.
Preliminary experiments investigating the interaction between EWS-FLI-1 and the REST promoter using the chromatin immunoprecipitation assay indicated that EWS-FLI-1 may bind to the REST promoter (data not shown), suggesting that EWS-FLI-1 directly regulates REST expression. However, further studies are needed to confirm these findings.
Inhibition of EWS-FLI-1 expression in TC71 and A4573 cells resulted in a reduction in REST protein expression (Fig. 2C). By contrast, altering the expression of REST in TC71 cells appeared to have no effect on EWS-FLI-1 expression. Taken together, these results indicate that REST is a downstream target of EWS-FLI-1. Although EWS-FLI-1 is known to control the expression of many other genes, we believe the current study data demonstrate for the first time that REST is regulated by EWS-FLI-1. Therefore, REST overexpression may contribute to the pathogenesis of Ewing sarcoma. The importance of REST to the growth and development of Ewing sarcoma was demonstrated by creating TC71-siREST tumor cell clones, injecting these cells into nude mice, and comparing their growth in vivo with that of siControl-transfected cells. Inhibition of REST resulted in significant decreased tumor growth (Fig. 3). Because we found no alteration in EWS-FLI-1 in TC71-siREST cells, the decreased tumor growth was not believed to be secondary to the downregulation of EWS-FLI-1, which has previously been reported. We also determined the role of REST on cell proliferation in vitro and in vivo. The results indicated that inhibition of REST did not significantly inhibit cell growth (data not shown), and therefore the reduction in tumor growth by REST inhibition does not appear to be due to a direct effect on tumor cell proliferation. We previously demonstrated that Ewing sarcoma is a vascular-rich tumor and that interference with vascular formation and tumor vessel expansion severely retards tumor growth and metastasis to the lung.[15, 17] We further demonstrated that VEGF165 and DLL4 are critical mediators of Ewing tumor vascular development and that pericytes play an important role in maintaining tumor oxygenation and tumor vessel blood flow. In the current study, we investigated whether the inhibition of REST affected tumor vessel characteristics and function. Neither CD31 nor VEGF expression were found to be altered in TC71-siREST tumors (Fig. 4A). However, there was a significant decrease observed in tumor vessel pericyte coverage. These findings suggest that tumor vessel morphology was altered in tumors in which REST expression was inhibited. Pericyte coverage of these vessels was severely compromised (Fig. 4D). This decrease in tumor vessel pericyte coverage correlated with increased tumor hypoxia and tumor cell apoptosis (Fig. 5). Pericytes are important for vascular stabilization, contribute to efficient vascular blood flow, and prevent vascular leakage.[20, 21] Decreased pericyte coverage results in decreased vascular proficiency, as characterized by increased vessel leakage and decreased tumor perfusion, resulting in tumor hypoxia. The results of the current study confirmed that decreased numbers of pericytes surrounding the tumor vasculature result in increased hypoxia and apoptosis, suggesting that REST controls pericyte coverage of tumor vessels and that inhibition of REST leads to decreased pericyte coverage and poor tumor perfusion, which results in increased hypoxia and tumor cell apoptosis.
We previously determined that the Notch pathway, specifically DLL4, regulates the differentiation of pericytes from bone marrow cells in patients with Ewing sarcoma. We demonstrated that DLL4 plays a crucial role in the formation of the pericyte layer surrounding tumor vessels and in the tumor vessel expansion required for growth. This pathway controlling pericyte development involves Hes1, one of the downstream effectors of the DLL4 pathway. In this study, DLL4 and Hes1 levels were found to be significantly reduced after the inhibition of REST (Fig. 6), confirming the correlation between the DLL4-Notch signaling pathway and vascular pericyte coverage. To the best of our knowledge, the current study is the first to demonstrate that REST and EWS-FLI-1 (Fig. 6D) activate the DLL4-Notch pathway in patients with Ewing sarcoma.
The data from the current study support the hypothesis that REST, a neuronal repressor gene, is upregulated by EWS-FLI-1 in patients with Ewing sarcoma and that increased REST expression contributes to the growth of the tumor. These findings suggest that REST is involved in tumor vascular expansion through a mechanism involving DLL4 and Hes1. Suppression of REST resulted in the inhibition of tumor growth, decreased levels of tumor vessel pericytes, and increased hypoxia and apoptosis. The finding that REST controls the formation of tumor vessels, vascular morphology, and function is novel. The results support our hypothesis that EWS-FLI-1, which is responsible for the malignant phenotype of Ewing sarcoma, controls the development of tumor vessels as well as the tumor vessel morphology. This regulation of tumor vasculature by EWS-FLI-1 is mediated in part by REST. Targeting of angiogenic pathways and disruption of the development of functional tumor vessels inhibited Ewing sarcoma growth in several mouse xenograft models.[17, 22, 23] A recent preliminary clinical trial indicated that the addition of low-dose antiangiogenic chemotherapy to standard multiagent chemotherapy may increase therapeutic benefit for patients with newly diagnosed metastatic Ewing sarcoma in the lung. Taken together, these data suggest that REST may be a potential therapeutic target among patients with Ewing sarcoma.
Supported by National Institutes of Health grant CA 103986 and core grant CA106672.