Fig. 1
Systems biology identifies TCF7L1 as a prognostically relevant EWSR1-ETS-regulated network hub whose re-expression inhibits tumorigenesis in vitro and in vivo. a) Workflow depicting a systems biology approach to identify DEGs regulated by EWSR1-ETS, involved in regulation of cell differentiation, functioning as highly interconnected TFs, and associated with overall survival in a cohort of 166 EwS patients. Number of genes represent remaining candidates after each filtering step. b) Network of EWSR1-ETS-regulated genes involved in regulation of cell differentiation. Genes are depicted as nodes (circles). Blue nodes represent TFs, node outline color show up- (green) or down- (red) regulation by EWSR1-ETS. Node border width represent strength of regulation by EWSR1-ETS (the thicker the border the higher the fold-change). Black dots surrounding the nodes represent interconnections with other nodes within the network. Connecting lines show three types of interconnection: physical (red), pathway (blue), genetic (brown). Line width represents strength of interconnection. c) Close-up image of highly interconnected TFs located at the center of the network (blue nodes). d) Kaplan-Meier survival analysis of 166 primary EwS patients stratified by quintile TCF7L1 expression. Mantel-Haenszel test, Bonferroni corrected. DEG: differentially expressed genes; KD: knockdown. e) Kaplan–Meier survival analysis of 114 EwS patients stratified by TCF7L1 protein expression intensity (low intensity≤1, high intensity> 1). P-value was determined by Gehan-Creslow-Wilcoxon test. Representative micrographs of TCF7L1 IHC are depicted. Scale bar = 50 μm. f) TCF7L1 expression in cell lines or primary tumors from EwS and 17 additional tumor entities (Cancer Cell Line Encyclopedia, CCLE). Data are represented as bar plots where horizontal bars represent mean and SEM. The number of samples per group (n) is given in parentheses. RMS, rhabdomyosarcoma; NSCLC, non-small cell lung carcinoma; SCLC: small-cell lung carcinoma. g) Weighted Gene Correlation Network Analysis (WGCNA) of enriched gene-sets obtained by Pearson correlation analysis of genes whose expression is negatively correlated with TCF7L1 expression in Affymetrix expression data of 166 EwS tumors. Network depicts signatures presenting NES > 1.5 and P < 0,05. NES, normalized enrichment score. h) Relative TCF7L1 expression (qRT-PCR) of TC-71 and SK-N-MC cells containing a DOX-inducible re-expression construct for TCF7L1. Cells were grown either with or without DOX for 72 h. n ≥ 6 biologically independent experiments. Two-sided Mann-Whitney test. i) Viable cell count of TC-71 and SK-N-MC cells containing a DOX-inducible TCF7L1 re-expression construct 72 h after DOX-treatment. Data are mean and SEM, n = 7 biologically independent experiments. Two-sided Mann-Whitney test. j) Relative area covered by colonies (%) grown in soft-agar of EwS cells containing a DOX-inducible re-expression construct for TCF7L1. Cells were grown either with/without DOX-treatment. n = 3 biologically independent experiments. Two-sided Mann-Whitney test. k) Growth of subcutaneous xenografts of indicated EwS cells containing a DOX-inducible TCF7L1 re-expression construct (arrow = start of DOX-treatment). Data are represented as means (n = 7 animals/group). Two-sided Mann-Whitney test. l) Ex vivo analysis of relative necrotic area (top) and mitotic index (bottom) of xenografted TC-71 and SK-N-MC cell lines. Data are mean and SEM, n = 7 animals/group. m) Ex vivo analysis of Ki67 positivity of xenografted TC-71 and SK-N-MC cell lines. Horizontal bars represent means and whiskers SEM, n = 7 animals/group. P-values were determined via χ2 test testing all positives (high and moderate immunoreactivity) versus negatives. Histological images depict representative Ki67 micrographs. Scale bar = 50 μm.