The transcriptional network for mesenchymal transformation of brain tumours

Abstract

The inference of transcriptional networks that regulate transitions into physiological or pathological cellular states remains a central challenge in systems biology. A mesenchymal phenotype is the hallmark of tumour aggressiveness in human malignant glioma, but the regulatory programs responsible for implementing the associated molecular signature are largely unknown. Here we show that reverse-engineering and an unbiased interrogation of a glioma-specific regulatory network reveal the transcriptional module that activates expression of mesenchymal genes in malignant glioma. Two transcription factors (C/EBPbeta and STAT3) emerge as synergistic initiators and master regulators of mesenchymal transformation. Ectopic co-expression of C/EBPbeta and STAT3 reprograms neural stem cells along the aberrant mesenchymal lineage, whereas elimination of the two factors in glioma cells leads to collapse of the mesenchymal signature and reduces tumour aggressiveness. In human glioma, expression of C/EBPbeta and STAT3 correlates with mesenchymal differentiation and predicts poor clinical outcome. These results show that the activation of a small regulatory module is necessary and sufficient to initiate and maintain an aberrant phenotypic state in cancer cells.

Figure 1. The mesenchymal signature of HGGs is controlled by six TFs

a, TFs involved in activation of MGES targets are shown in pink, those involved in repression are in purple. MGES targets controlled by these TFs are in cyan. Overall, the six TFs control 74% of the genes in the mesenchymal signature of high-grade glioma. A region between 2 kb upstream and downstream the transcription start site of the target genes identified by ARACNe was analyzed for the presence of putative binding sites. Genomic regions of genes containing putative binding sites for specific TFs were immunoprecipitated in SNB75 cells by antibodies specific for b, Stat3; c, C/EBPβ; d, FosL2; e, bHLH-B2. SOCS3 was included as positive control of Stat3 binding. Total chromatin before immunoprecipitation was used as positive control for PCR. The OLR1 gene was used as negative control. f, Summary of binding results of the tested TFs to mesenchymal targets.

Figure 2. A hierarchical transcriptional module regulates the MGES

ChIP for a, C/EBPβ; b, Stat3. c, Transcriptional network emerging from promoter occupancy analysis. d, qRT-PCR of mesenchymal TFs in glioma cells infected with Stat3/C/EBPβ shRNA or controls lentiviruses. e, Venn-diagram depicts the proportion of mesenchymal genes identified by ARACNe as targets of only C/EBPβ, Stat3 or both TFs. f, Heatmap of MGES gene expression analysis of mouse and human cells carrying perturbations of C/EBPβ plus Stat3. Samples (columns) were grouped according to species and treatment. Control, control shRNA or empty vector; S-, Stat3 knock-down; S+, Stat3 overexpression; C-, C/EBPβ knock-down; C+, C/EBPβ overexpression; S-/C-, Stat3/ C/EBPβ knockdown; S+/C+, Stat3/ C/EBPβ overexpression. g, GSEA of the MGES on the gene expression profile rank-sorted according to the correlation with the C/EBPβxStat3 metagene. The bar-code plot indicates the position of MGES genes, red and blue colors represent positive and negative correlation, respectively. The gray scale bar indicates the spearman rho coefficient used as weighting score for GSEA. nES, normalized enrichment score; p, sample-permutation-based p-value.

Figure 3. Ectopic expression of C/EBPβ and Stat3C in NSCs induces mesenchymal transformation and inhibits neural differentiation

a, Immunofluorescence analysis for SMA and fibronectin in C17.2 expressing the indicated TFs. b, qRT-PCR of mesenchymal targets in C17.2 expressing the indicated TFs. n = 3; Bars indicate Mean±SD. c, Alcian blue staining of C17.2 expressing Stat3C and C/EBPβ or the empty vector cultured in growth medium (upper panels), or chondrocyte differentiation medium (lower panels). d, Microphotographs of invading C17.2 expressing Stat3C and C/EBPβ or empty vector. e, Quantification of invading cell in the absence or in the presence of PDGF. n = 3; Bars indicate Mean±SEM. f, Immunofluorescence analysis for CTGF in NSCs expressing Stat3C and C/EBPβ or the empty vector. GFP identifies infected cells. g, Quantification of GFP⁺/CTGF⁺ cells. Bars indicate Mean±SD of three independent experiments. h, qRT-PCR of representative mesenchymal genes in primary NSCs expressing the indicated TFs. n = 3; Bars indicate Mean±SD. i, qRT-PCR of βIII-tubulin, doublecortin and GFAP in NSCs expressing Stat3C plus C/EBPβ or the empty vector. n= 3; Bars indicate Mean±SD. qRT-PCR data are 18S ribosomal RNA normalized fold changes. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 4. C/EBPβ and Stat3 maintain the mesenchymal phenotype of human glioma cells

a, Immunofluorescence for fibronectin, Col5A1 and YKL40 in BTSC-3408 infected with lentiviruses expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. b, Quantification of fibronectin; c, Col5A1; and d, YKL40 positive cells. n = 3 independent experiments; Bars indicate Mean±SD. e, qRT-PCR of mesenchymal genes in BTSC-20 infected with lentiviruses expressing Stat3, C/EBPβ, or Stat3 plus C/EBPβ shRNA. Gene expression was normalized to the expression of 18S ribosomal RNA. n = 3; Bars indicate Mean±SD. f, Microphotograps of invading SNB19 cells infected with lentiviral vectors expressing control or shStat3 plus shC/EBPβ. g, Quantification of SNB19 invading cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). h, Invading BTSC-3408 cells infected with shCtr, shStat3, shC/EBPβ or shStat3 plus shC/EBPβ lentiviruses. i, Quantification of invading BTSC-3408 cells. Bars indicate Mean±SD; n = 6 (two independent experiments, each performed in triplicate). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 5. C/EBPβ and Stat3 are essential for glioma tumor aggressiveness in mice and humans

a, Immunofluorescence staining for human vimentin, CD31, fibronectin, Col5A1, and YKL40 in tumors derived from SNB19 cells infected with lentiviruses expressing shRNA targeting Stat3, C/EBPβ, or Stat3 plus C/EBPβ. T, tumor; B, normal brain. b, Kaplan-Meier survival curve of NOD SCID mice transplanted intracranially with SNB19 glioma cells transduced with shCtr (red), shStat3 (black), shC/EBPβ (green) or shStat3 plus shC/EBPβ (blue) lentiviruses. c, Immunostaining for human vimentin and Ki67 on representative brain sections from mice injected with BTSC-3408 after silencing of C/EBPβ and Stat3. St, striatum; CC, corpus callosum. d, Quantification of human vimentin positive area. e, Quantification of Ki67 positive cells. n=5 for each group; Bars indicate Mean±SD. f, Immunostaining for fibronectin and g, Col5A1 on representative brain sections from mice injected with BTSC-3408 transduced as indicated. h, Kaplan-Meier analysis comparing survival of patients carrying tumors double positive for C/EBPβ and Stat3 (red) and single or double negative tumors (black). *p ≤ 0.05; **p ≤ 0.01.