Fig. 3

ASCL1 directly represses the expression of the MES gene NDRG1. a CSCs and GCLs show distinct patterns of endogenous ASCL1, NDRG1, and EGFR expression (WB). b The gene signature associated with high NDRG1 expression in PN CSC lines is enriched in the PN subgroup but also comprises a subset of genes (black arrow) correlated with the MES subgroup (GSEA). c ASCL1, NDRG1, and EGFR expression is upregulated in the human PN, MES, and P/CL GBM subgroups, respectively (upper histograms, expression data from Phillips dataset; lower histograms, expression data from the TCGA). d The expression and activation of NDRG1 by IHC are restricted to GBM specimens classified as MES (NDRG1 and pNDRG1, brown). e High ASCL1 expression in GBM positively correlates with a slightly more favorable prognosis than low expression, whereas high NDRG1 expression is associated with a worse outcome (Kaplan–Meier survival curves; data from TCGA). No differences in survival are retrieved in patients stratified based on EGFR expression. f ASCL1 overexpression in CSCs downregulates NDRG1 and EGFR expression (size-based WB). g The same results were confirmed by charge-based WB (Representative CSC lines: L0605 and L0512). h ASCL1 silencing de-represses NDRG1 and EGFR expression (R7epresentative CSC line: L0627). i Genomic snapshot depicting NDRG1 transcription start site and its promoter region. ASCL1 binding sites are indicated by solid black boxes. Representative ASCL1 ChIP-seq data produced in human GBM CSCs (red profile) [29] and in multiple lung cancer cell lines from independent laboratories (blue and black profiles) [28, 30] are reported. The yellow highlight indicates the site screened by ChIP-qPCR in L0512 and L0605 CSC lines. Coverage data are represented as normalized RPM. (j) ChIP analysis indicates binding of ASCL1 to promoter/intragenic binding sites of NDRG1 and EGFR genes