Fig. 1.
CRE is a pan-cancer TME responsive stemness effector. a A scheme to screen TME responsive transcriptional regulators of CSCs. Gene set enrichment analysis (GSEA) in paired sphere-adherent cells identifies CSC-enriched transcription factors. Transcription factor motif analysis of genes enriched in StemnessScorehigh tumors identified TME responsive stemness factors in vivo. The STRING (http://string-db.org/) interaction network of 31 TME responsive stemness transcription factors overlapped in GSEA/TCGA datasets was present in the right panel. CRE associated factors were labeled in red in the network. b Normalized enrichment scores (NES) for top transcription factors enriched in 17 paired sphere-adherent from 15 independent datasets. NES was determined by GSEA. CRE, cAMP responsive element. c Sphere formation of sorted cells (MDA-MB-231, MCF-7, DLD1 and T47D) according to the CRE-responsive GFP reporter (CRE-dGFP) activity (n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA). d In vitro limit dilution assay of sorted CRE-dGFP+ and CRE-dGFP- populations in MDA-MB-231 and T47D cells. Differences in stem cell frequencies were determined by ELDA (https://bioinf.wehi.edu.au/software/elda/). n = 6 for each group. e Primary (left panel) and secondary (right panel) sphere formation of MDA-MB-231 cells in indicated doses of cAMP mimics in sphere media (Butyl-cAMP and 8-Br-cAMP, n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA). f Western blot analysis of MDA-MB-231 (upper panel) and DLD1 cells treated with PKA inhibitor H89 for 1 hr. Cell extracts were analyzed with antibodies against phospho-CREB1/ATF1, total ATF1/CREB1 and alpha-tubulin. g Representative images of sphere formation in MDA-MB-231 (left panel) and DLD1 (right panel) cells treated with H89 in sphere media. Scale bar represents 60 μm. h Primary and secondary sphere formation of MDA-MB-231 (left panel) and DLD1 (right panel) cells treated with H89 in sphere media (n = 3; mean ± SD; P values, Tukey’s multiple comparisons after 1-way ANOVA). i The prognostic meta-z sores of ATF1/CREB1 target genes (collected form MSigDB, Supplementary Table S2) among cancer types. Meta-z scores were calculated by unweighted prognostic z scores of individual genes of the signature. j Representative IHC staining of phospho-CREB1/ATF1 in breast cancer specimens. Numbers indicate phospho-CREB1/ATF1 expression scores. Scale bar represents 50 μm. k Kaplan Meier curves of estimated overall survival (OS, left panel) and disease-free survival (DFS, right panel) of breast cancer patients with low (n = 238) and high (n = 128) phospho-CREB1/ATF1 levels (P values, log-rank test). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant