Fig. 5: The +1q is associated with increased Notch activation in vitro and in human tumors due to increased γ-secretase gene dosage on 1q.
From: Chromosome evolution screens recapitulate tissue-specific tumor aneuploidy patterns
a, Hallmark GSEA profile clustering of +1q or +8q HMECs and breast cancers relative to WT counterparts. MYC and Notch gene sets outlined in black. ROS, reactive oxygen species; met., metabolism; DN, down; UPR, unfolded protein response; Inflam., inflammatory; EMT, epithelial-to-mesenchymal transition; Ox phos, oxidative phosphorylation; UV, ultraviolet. b, Diagram of ligand-based Notch activation assay. DLL, DLL1 + DLL4 recombinant protein. c, Curated Notch activation and repression gene set enrichment in HMECs after 20 h ligand exposure. GSEA scores (0.80 and −0.75) and associated P values (P = 5.7 × 10−4 and P = 0.001) are shown, respectively, for Notch activation (up) and repression (DN) sets. d, Signed −log10 P values from GSEA with curated Notch gene sets for +1q versus WT 1q differential expression rankings across tumor types, sorted by prevalence of +1q. Cancer types with <10 samples in the CCLE77 not included (hatched squares). DN, down. e, Western blots (top) showing cleaved NOTCH1 (N1ICD) in +1q and WT 1q HMEC lines (bottom) in response to calcium depletion (4 mM EGTA, 10 min), ±GSI (5 μM L-685,458, pre-incubated for 30 min). GAPDH shown as loading control. f, Quantification of N1ICD (e), normalized to GAPDH. P values calculated from two-sided Wilcoxon test. g, Diagram of the γ-secretase complex and gene locations on 1q. h, Ranked 1q-resident gene mRNA/DNA correlations (signed −log10 P value from Pearson’s correlations) in matched tumor/normal BRCA samples. γ-secretase genes labeled red. Other proposed drivers of 1q are also shown. i,j, Western blot quantification of NCSTN protein (i) and N1ICD (j) in diploid (WT 1q) and +1q HMECs infected with lentivirus containing either control (sgAAVS1) or NCSTN-targeting CRISPR guides and treated with EGTA for 10 min. NCSTN and N1ICD levels were normalized to GAPDH, and NCSTN/GAPDH and N1ICD/GAPDH ratios were normalized to the average ratio of sgAAVS1 WT 1q cells. P values were calculated from two-sided t-test, not corrected for multiple testing. n.s., not significant. k, Correlation between NCSTN and N1ICD protein levels in each sample quantified in i and j. Pearson’s correlation coefficient squared (r2 = 0.73) and associated P value (P = 1.15 × 10−7) are shown. Dashed line indicates linear regression model of the data.
