Fig. 6: SNAIL overcomes senescence and the p16INK4A cell cycle restriction checkpoint without altering the Trp53/p21CIP1 axis.
From: Non-canonical functions of SNAIL drive context-specific cancer progression
a, b Representative Trp53 and p21CIP1 stainings of PanINs (a) and PDAC (b) of PKrasG12D/+;SnailKI/+ animals (n = 3 each). c Western blot of TRP53 and p21CIP1 expression in PKrasG12D/+;SnailKi/+ (n = 3) and PKrasG12D/+ (n = 1) PDAC cell lines after 6 h 20 μM etoposide (Eto) or vehicle (DMSO) treatment. d GSEA of mRNA expression of 1-month-old mice (n = 2 per genotype) computed and corrected for multiple testing using the Benjamini–Hochberg procedure (for statistical details, see methods section) shows significant enrichment of KEGG p53 signalling pathway genes in PKrasG12D/+;SnailKI/+ (red) vs. PKrasG12D/+ (blue). Normalized Enrichment Score: 1.98; Nominal p-value < 0.001; False Discovery Rate (FDR) q-value < 0.001. e, f Kaplan-Meier survival curves of PKrasG12D/+ (n = 125; 465 days), PKrasG12D/+;SnailKI/+ (n = 42; 190 days), PKrasG12D/+;Trp53R172H/+ (n = 28; 117 days) and PKrasG12D/+;SnailKI/+;Trp53R172H/+ (n = 22; 90 days) animals. ***p < 0.0001, log-rank test with Bonferroni correction. g Representative p16INK4A staining of PanINs (left) and PDAC (right) of PKrasG12D/+;SnailKI/+ mice (n = 3 each). h qRT-PCR analysis of p16Ink4a (left) and p19Arf (right) mRNA expression in PDAC of endpoint mice (PKrasG12D/+ n = 5; PKrasG12D/+;SnailKI/+ n = 12; PKrasG12D/+;SnailKI/KI n = 12). Mean ± SEM, **p = 0.0094, *p = 0.0365, Mann-Whitney two-tailed test. i Scheme of Cdkn2a gene locus and p16Ink4a genotyping strategy. The non-related proteins p16INK4A and p19ARF are encoded both by the Cdkn2a locus. Red arrows, primer positions. UTR, untranslated region. Scheme according to35. j PCR of p16Ink4a genomic sequence integrity in PDAC cell lines of PKrasG12D/+ (n = 30), PKrasG12D/+;SnailKI/+ (n = 13); PKrasG12D/+;SnailKI/KI (n = 9) endpoint mice. Gabra, internal positive control. k Quantification of PCR analysis of p16Ink4a genomic sequence integrity of data in panel (j). **p = 0.0016, two-tailed Fisher’s exact test. l Kaplan-Meier survival curves of PKrasG12D/+;SnailKI/+ (n = 42; 190 days), PKrasG12D/+;SnailKI/+;p16Ink4a*/+ (n = 22; 156 days) and PKrasG12D/+;SnailKI/+;Cdkn2alox/+ with loss of p16INK4A and p19ARF (n = 17; 108 days). ***p < 0.0001, log-rank test with Bonferroni correction. Note: PKrasG12D/+ cohort in panel (e, f) is the same shown in Figs. 1g, 4b, and PKrasG12D/+;SnailKI/+ cohort of panel e and l is the same shown in Fig. 1g. Source data of Fig. 6 are provided in the Source Data file. Scale bars, 50 μm. ns, not significant.
