Journal: Genes & Development

Article Title: Mutant p53 regulates Survivin to foster lung metastasis

doi: 10.1101/gad.340505.120

Figure Lengend Snippet: Mutant p53 promotes ESCC invasion and lung metastasis. ( A , left ) Trp53 R172H is depleted by shRNA in Trp53 R172H/− cells. shRNA-resistant Trp53 R172H is reintroduced to Trp53 R172H/− shTrp53 ( middle ), and Trp53 R172H is ectopically expressed in Trp53 −/− cells ( right ). Relative intensity densitometry of TRP53 results are shown at the bottom . ( B ) Depletion of Trp53 R172H suppresses cell invasion, whereas reintroduction of shRNA-resistant Trp53 R172H or ectopic expression of Trp53 R172H promotes cell invasion in a Boyden chamber assay. Trp53 R172H/− shCtrl versus shTrp53, n = 4 per group, P = 0.0035, unpaired t -test; Trp53 R172H/− shRNA + Ctl versus + Trp53 R172H , n = 3 per group, P = 0.0398, unpaired t -test; Trp53 −/− Ctrl versus R172H, n = 3 per group, P = 0.0094, unpaired t -test. Error bars represent SEM. ( C , D ) YFP+ ESCC cells were injected into the tail vein to induce lung metastatic nodules. Mice were sacrificed 8 wk after injection. Lung metastasis colony number in Trp53 R172H/− shCtrl injected mice was increased greatly compared with shTrp53 injected mice. Trp53 R172H/− shCtrl versus shTrp53, n = 3 per group, P < 0.0001, unpaired t -test. Scale bar, 1 mm. ( E , F ) YFP+ Trp53 R172H/− shTrp53 cells reintroduced with shRNA-resistant Trp53 R172H were used to repeat the tail-vein injection assay. Mice were sacrificed 8 wk after injection. Trp53 R172H/− shTrp53-rescued cell line-injected mice had a trend of increased lung metastatic colony number. Trp53 R172H/− shTrp53+Ctrl ( n = 4 per group) versus shTrp53+ Trp53 R172H ( n = 3 per group), P = 0.0709, unpaired t -test. Scale bar, 1 mm.

Article Snippet: Alternatively, CRISPR/Cas9 against Trp53 (pLentiCRISPR v2, GenScript) or an empty vector control were used for infection, and puromycin selection and Trp53 expression validation were performed as described.

Techniques: Mutagenesis, shRNA, Expressing, Boyden Chamber Assay, Injection

Journal: Genes & Development

Article Title: Mutant p53 regulates Survivin to foster lung metastasis

doi: 10.1101/gad.340505.120

Figure Lengend Snippet: Depletion of Trp53 R172H in ESCC cells alters the transcriptome during lung metastasis. ( A ) Depletion of Trp53 R172H is maintained in isolated metastatic cell lines. Relative intensity densitometry of TRP53 results are shown at the bottom . ( B ) Principle component analysis (PCA) demonstrated distinct gene expression profiles depending upon p53 status and primary tumor versus metastatic tumor status. ( C ) Heat map of differentially regulated genes upon Trp53 R172H depletion in primary and metastatic cells, profiled by RNA-seq. ( D ) Volcano plot of differentially regulated genes in metastatic cells. ( E ) Numbers of significantly ( P adj < 0.01, |log 2 FC| > 2) changed genes in metastatic Trp53 R172H/− cells ( n = 5) compared with Trp53 −/− cells ( n = 3). ( F ) GSEA reveals hallmark pathways significantly enriched in shCtrl-M and shTrp53-M cells. (M) Metastatic.

Article Snippet: Alternatively, CRISPR/Cas9 against Trp53 (pLentiCRISPR v2, GenScript) or an empty vector control were used for infection, and puromycin selection and Trp53 expression validation were performed as described.

Techniques: Isolation, Gene Expression, RNA Sequencing

Journal: Genes & Development

Article Title: Mutant p53 regulates Survivin to foster lung metastasis

doi: 10.1101/gad.340505.120

Figure Lengend Snippet: The expression of SURVIVIN depends upon Trp53 R172H status. ( A ) GSEA reveals enrichment of the YAP signature in Trp53 R172H/− shCtrl-M cells. ( B ) RNA-seq shows that BIRC5 , a YAP target gene, is increased significantly in Trp53 R172H/− shCtrl-M cells. ( C ) QRT-PCR of BIRC5 confirms its up-regulation in Trp53 R172H/− shCtrl-M compared with shTrp53-M cells. Trp53 R172H/− shCtrl-M ( n = 5) versus shTrp53-M ( n = 3), P = 0.0287, unpaired t -test. Error bars represent SEM. ( D ) Immunohistochemical staining of SURVIVIN in 3D organoids form by Trp53 R172H/− shCtrl and shTrp53 cells. Trp53 R172H/− shCtrl ( n = 10 organoids) versus shTrp53 ( n = 14 organoids), P = 0.016, unpaired t -test. Error bars represent SEM. Scale bar, 100 µm. ( E ) Immunohistochemical staining of p53 ( top ) and SURVIVIN ( bottom ) in the esophagus epithelium of Trp53 R172H/− and Trp53 −/− mice treated with 4NQO. P53 staining: Trp53 R172H/− versus Trp53 −/− , n = 8 each group. (*) P = 0.0168, unpaired t -test. SURVIVIN staining: Trp53 R172H/− versus Trp53 −/− , n = 8 each group. Error bars represent SEM. Scale bar, 100 µm. ( F ) Immunohistochemical staining of SURVIVIN in Trp53 R172H/− shCtrl and shTrp53 in metastatic lung lesions. Trp53 R172H/− shCtrl ( n = 15 tumors) versus shTrp53 ( n = 10 tumors), P = 0.0178, unpaired t -test. Error bars represent SEM. Scale bar, 100 µm.

Article Snippet: Alternatively, CRISPR/Cas9 against Trp53 (pLentiCRISPR v2, GenScript) or an empty vector control were used for infection, and puromycin selection and Trp53 expression validation were performed as described.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Genes & Development

Article Title: Mutant p53 regulates Survivin to foster lung metastasis

doi: 10.1101/gad.340505.120

Figure Lengend Snippet: Depletion of BIRC5 attenuates Trp53 R172H -driven lung metastasis. ( A , B ) SURVIVIN expression is depleted or decreased significantly in Trp53 R172H/− or Trp53 −/− cells with two independent shRNAs (shBIRC5.1 and shBIRC5.2). Relative intensity densitometry of SURVIVIN results are shown at bottom . ( C , D ) BIRC5 depletion in Trp53 R172H/− or Trp53 −/− cells is confirmed by qPCR. Trp53 R172H/− BIRC5 Ctrl versus shBIRC5.1 versus shBIRC5.2, n = 3 per group, (**) P = 0.0021 Trp53 R172H/− BIRC5 Ctrl versus shBIRC5.1, (**) P = 0.0015 Trp53 R172H/− BIRC5 Ctrl versus shBIRC5.2, one-way ANOVA. Trp53 −/− BIRC5 Ctrl versus shBIRC5.1 versus shBIRC5.2, n = 3 per group. Error bars represent SEM. ( E , F ) Depletion of BIRC5 significantly decreased cell invasion in Trp53 R172H/− but not Trp53 −/− cells. Trp53 R172H/− BIRC5 Ctrl versus shBIRC5.1 versus shBIRC5.2, n = 9 per group, (****) P < 0.0001 Trp53 R172H/− BIRC5 Ctrl versus shBIRC5.1, (****) P < 0.0001 Trp53 R172H/− BIRC5 Ctrl versus shBIRC5.2, one-way ANOVA. Error bars represent SEM. ( G , H ) BIRC5 depletion significantly reduces subcutaneous tumor growth at early stage. Trp53 R172H/− BIRC5 Ctrl ( n = 6 flanks) versus shBIRC5.1 ( n = 8 flanks) versus shBIRC5.2 ( n = 6 flanks). (**) P = 0.0042 day 8; (*) P = 0.00214 day 11; (***) P = 0.0004 day 13; (*) P = 0.0261 day 17, one-way ANOVA. Error bars represent SEM. Trp53 −/− BIRC5 Ctrl ( n = 6 flanks) versus shBIRC5.1 ( n = 6 flanks) versus shBIRC5.2 ( n = 6 flanks). (*) P = 0.0322 day 10; (**) P = 0.0016 day 12, one-way ANOVA. Error bars represent SEM. ( I ) Depletion of BIRC5 in Trp53 R172H/− ESCC cells significantly reduces lung metastasis burden number detected at week 8 after tail-vein injection. BIRC5 Ctrl ( n = 6) versus shBIRC5.1 ( n = 5), P = 0.0057; BIRC5 Ctrl ( n = 6) versus shBIRC5.2 ( n = 4), P = 0.0009, one-way ANOVA. Error bars represent SEM. Scale bar, 1 mm. ( J ) Depletion of BIRC5 in Trp53 −/− ESCC cells does not significantly reduce lung metastasis burden number detected at week 8 after tail-vein injection. BIRC5 Ctrl ( n = 4) versus shBIRC5.1 ( n = 3) versus shBIRC5.2 ( n = 4). Error bars represent SEM. Scale bar, 1mm.

Article Snippet: Alternatively, CRISPR/Cas9 against Trp53 (pLentiCRISPR v2, GenScript) or an empty vector control were used for infection, and puromycin selection and Trp53 expression validation were performed as described.

Techniques: Expressing, Injection

Journal: Genes & Development

Article Title: Mutant p53 regulates Survivin to foster lung metastasis

doi: 10.1101/gad.340505.120

Figure Lengend Snippet: Trp53 R172H , not wild-type p53, binds to YAP and fosters Survivin expression in murine ESCC cells. ( A , left ) Endogenous expression level of YAP and p53 in wild type, Trp53 R172H/− shCtrl, shTrp53, Trp53 −/− Ctrl, and Trp53 −/− R172H cells. ( Right ) Expression levels of YAP and p53 in LS123 nontarget control (NT), Crispr knockout (Crispr) cell lines, and H1299 empty vector control (control) and p53 R175H (R175H) ectopic expressed cell lines. ( B ) Proximity ligation assay (PLA) for YAP and Trp53 R172H . Scale bar, 50 µm. ( C ) Quantification of the average number of PLA signals shows significantly high levels of signals in Trp53 R172H/− shCtrl cells (vs. shTrp53 cells, n > 100 cells per group, P < 0.0001, unpaired t -test, error bars represent SEM) and Trp53 −/− R172H cells (vs. Ctrl cells, n > 100 cells per group, P < 0.0001, unpaired t -test, error bars represent SEM). ( D ) Immunoprecipitation-Western blot analysis of Trp53 R172H -HA with YAP-DDK in 293 FT cells. ( E ) Chromatin immunoprecipitation (ChIP) with YAP antibody in WT1, Trp53 R172H/− shCtrl, and shTrp53 cells demonstrates increased level of YAP on the BIRC5 promoter in the presence of Trp53 R172H . shCtrl versus WT1 cells, n = 3 replicates per group, P = 0.0052, unpaired t -test; shCtrl versus shTrp53 cells, n = 3 replicates per group, P = 0.0107, unpaired t -test. Error bars represent SEM. ( F ) Proximity ligation assay for YAP and p53 R175H . Scale bar, 20 µm. ( G ) Quantification of the average number of PLA signals shows significantly high levels of signals in LS123 NT cells (vs. Crispr cells, n > 100 cells per group, P < 0.0001, unpaired t -test, error bars represent SEM) and H1299 R175H cells (vs. control cells, n > 100 cells per group, P < 0.0001, unpaired t -test, error bars represent SEM).

Article Snippet: Alternatively, CRISPR/Cas9 against Trp53 (pLentiCRISPR v2, GenScript) or an empty vector control were used for infection, and puromycin selection and Trp53 expression validation were performed as described.

Techniques: Expressing, Control, CRISPR, Knock-Out, Plasmid Preparation, Proximity Ligation Assay, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation