ZEB2 Represses the Epithelial Phenotype and Facilitates Metastasis in Ewing Sarcoma

Abstract

The vast majority of cancer-related deaths are attributable to metastasis. Effective treatment of metastatic disease will be improved by a better understanding of the molecular mechanisms contributing to this phenomenon. Much of the work in this field has focused on metastasis of carcinomas, tumors of epithelial origin, while metastasis of sarcomas, tumors of mesenchymal origin, remains poorly understood. Experimental evidence from studies in carcinomas, coupled with clinical observations, highlights the importance of both epithelial and mesenchymal characteristics in these cancer cells that make them competent for metastasis. We set out to test if similar cellular plasticity contributes to sarcoma metastasis. We found that the transcription factor, ZEB2, repressed epithelial gene expression in Ewing sarcoma cells, and this, in turn, repressed the epithelial phenotype. When ZEB2 was experimentally reduced in these cells, epithelial characteristics including decreased migratory ability and cytoskeleton rearrangements were observed. Furthermore, ZEB2 reduction in Ewing sarcoma cells resulted in a decreased metastatic potential using a mouse metastasis model. Our data show that Ewing sarcoma cells may have more epithelial plasticity than previously appreciated. This coupled with previous data demonstrating Ewing sarcoma cells also have mesenchymal features primes these cells to successfully metastasize. This is clinically relevant for 2 important reasons. First, this may offer a therapeutic opportunity to induce characteristics of one cell type or the other depending on the stage of the disease. Second, and more broadly, this raises questions about the cell of origin in Ewing sarcoma and may inform future animal models of the disease.

Keywords: EMT; EWS/FLI; Ewing sarcoma; ZEB2; metastasis.

Figure 1.

ZEB2 is expressed in Ewing sarcoma cells. (A) Gene expression data from Ewing sarcoma patient tumors from the Baird et al. dataset showing the expression of epithelial markers, E-cadherin (CDH1), tight junction protein 1 (ZO-1), lamanin beta 1 (LAMB1), and mucin 1 (MUC1), mesenchymal markers, N-cadherin (CDH2), vimentin (VIM), fibronectin (FN), and integrin alpha 5 (ITGA5), and EMT-TF, Snail1 (SNAI1), Snail2/Slug (SNAI2), TWIST1, ZEB1 and ZEB2. Horizontal line indicates mean. (B) qRT-PCR data showing the expression of EMT-TF in a panel of Ewing sarcoma cell lines—A673, RDES, SKNMC, TC71, TC32, CHLA10, and CHLA258. (C) Western blot showing EWS/FLI and ZEB2 expression in control shRNA and EWS/FLI shRNA infected Ewing sarcoma cells lines, A673, SKNMC, and TC71. (D) qRT-PCR data showing EWS/FLI and ZEB2 expression levels in control shRNA and EWS/FLI shRNA infected Ewing sarcoma cell lines A673, SKNMC, and TC71. Errors bars represent standard deviation (SD) from 3 technical repeats. (E) ZEB2 gene expression in hBM-MSC and hNCSC from the von Levetzow et al. data set. Error bars represent SD.

Figure 2.

ZEB2 represses epithelial gene expression in Ewing sarcoma cells. (A) qRT-PCR data and western blot showing ZEB2 expression levels in A673 cells transfected with control siRNA or 2 different siRNAs targeting ZEB2 (siZEB2-5 and siZEB2-6). Tubulin is shown as a loading control. (B) Graph showing the top 10 enriched terms by DAVID analysis in the ZEB2 repressed gene set from our RNAseq analysis. Enrichment score equals −log(mean P-value). (C) Heat map and hierarchical clustering of the ZEB2 RNAseq data with Taube et al. EMT data in HMLE cells. (D) Heat map and hierarchical clustering of the ZEB2 RNAseq data set compared to the Onder et al. EMT data set in HMLE cells. (E) Heat map of the ZEB2 RNAseq data with Keshamouni et al. EMT time course data in A549 lung carcinoma cells. Color key represents log₂ FC.

Figure 3.

ZEB2 represses the epithelial phenotype in Ewing sarcoma cells. (A) qRT-PCR data showing ZEB2 knock-down in A673, SKNMC, and TC71 cells infected with control shRNA or shRNA targeting ZEB2 and the corresponding change of expression in the indicated genes. Error bars represent SD from technical triplicates. (B) Western blot showing expression of ZEB2, KRT8, and DSP in A673, SKNMC, and TC71 cells expressing a control or ZEB2-targeting shRNA. Tubulin is shown as a loading control. (C) Boyden chamber cell migration assays in A673, SKNMC, and TC71 cells infected with a control shRNA or ZEB2 targeting shRNA. Data are from 2 independent experiments each done in triplicate. Each point represents cells counted in one field—5 fields were counted per chamber. Significance was determined by Student’s t-test and the mean is shown. Horizontal bar represents mean. (D) Representative images from 3 time points of a wound healing assay using control or ZEB2 shRNA infected A673 and SKNMC cells (left). Scale bar = 250 µm. Quantification of the area of the wound healed measured every 24 hours (right). Data are the average of 2 independent experiments each done in triplicate (n = 6). P-values indicated above each condition as determined by Student’s t-test and error bars show SD. (E) Immunofluorescence images showing ZEB2 expression levels (red—568 nm) and actin cytoskeleton stained with phalloidin (green—488 nm) in A673 cells transfected with control siRNA or siRNA targeting ZEB2 (siZEB2-5). Scale bar = 50 µm.

Figure 4.

Expression of miR-200 family members epithelializes Ewing sarcoma cells. (A) qRT-PCR data showing the expression of the indicated miRNA in A673 and SKNMC cells infected with a lentivirus expressing a control miRNA or miR-200 family cluster from chromosome 1 (miR-200b, miR-200a, miR-429). Error bars represent SD from 3 technical repeats. (B) Western blot showing expression of ZEB2, KRT8, and DSP in A673 and SKNMC cells expressing a control miRNA or miR-200 family cluster from chromosome 1 (miR-200b, miR-200a, miR-429). Tubulin is shown as a loading control. (C) Representative images from 3 time points of a wound healing assay using control miRNA or miR-200 family cluster from chromosome 1 (miR-200b, miR-200a, miR-429) infected A673 and SKNMC cells (left). Scale bar = 250 µm. Quantification of the area of the wound healed measured every 24 hours (right). Data are the average of 2 independent experiments each done in triplicate (n = 6). P-values indicated above each condition as determined by Student’s t-test and error bars show SD.

Figure 5.

Reduction of ZEB2 decreases metastatic potential in Ewing sarcoma cells. (A) Volume of the right tibia from mice 6 weeks postinjection with either 25K or 100K control or ZEB2 shRNA (as indicated) infected A673 cells. Horizontal bar indicates mean. (B) Pie charts showing the total number of mice with macroscopic metastatic lung lesions at the time of sacrifice (6 weeks postinjection). Combined data includes both 25K and 100K conditions. (C) Images of lungs (front and back) from the animal from each condition bearing the highest metastatic burden. Scale bar = 5 mm. (D) Graph showing the percentage of the lung containing macroscopic pulmonary lesions in each condition, as indicated. P-value determined by Wilcoxon Mann–Whitney test. Horizontal bar indicates mean. (E) Correlation of metastatic burden (y axis) and tumor size (x axis). Pearson’s correlation coefficient for shControl = −.2892 and shZEB2 = −.4414. (F) Immunohistochemical staining for ZEB2 on lung metastases from indicated conditions. No primary antibody control is shown for control shRNA 25K sample’s adjacent section. Scale bar = 250 µm.

Figure 6.

ZEB2 levels correlate with patient metastasis. (A) ZEB2 expression levels determined by immunofluorescence intensity read by the AQUA system in tumor samples from patients with and without metastasis. Horizontal bar indicates the mean. P-value was determined by Student’s t-test. (B) Kaplin-Meier plot of overall patient survival. Patients were stratified based on ZEB2 expression levels—high ZEB2 represents values above the median and low ZEB2 represents values below the median.

Figure 7.

Model for ZEB2 in Ewing sarcoma. ZEB2 is expressed in Ewing sarcoma cells and acts to repress epithelial differentiation. This works in conjunction with EWS/FLI, which represses mesenchymal differentiation, to keep Ewing sarcoma cells in an undifferentiated state. Ewing sarcoma cells retain access to features of both epithelial and mesenchymal cells, which positions them for successful growth and metastasis.