Different hotspot p53 mutants exert distinct phenotypes and predict outcome of colorectal cancer patients

Abstract

The TP53 gene is mutated in approximately 60% of all colorectal cancer (CRC) cases. Over 20% of all TP53-mutated CRC tumors carry missense mutations at position R175 or R273. Here we report that CRC tumors harboring R273 mutations are more prone to progress to metastatic disease, with decreased survival, than those with R175 mutations. We identify a distinct transcriptional signature orchestrated by p53R273H, implicating activation of oncogenic signaling pathways and predicting worse outcome. These features are shared also with the hotspot mutants p53R248Q and p53R248W. p53R273H selectively promotes rapid CRC cell spreading, migration, invasion and metastasis. The transcriptional output of p53R273H is associated with preferential binding to regulatory elements of R273 signature genes. Thus, different TP53 missense mutations contribute differently to cancer progression. Elucidation of the differential impact of distinct TP53 mutations on disease features may make TP53 mutational information more actionable, holding potential for better precision-based medicine.

Fig. 1. TP53 R273 mutations in CRC are preferentially associated with more aggressive cancer features and shorter overall survival.

a Relative abundance of R175 and R273 TP53 hotspot mutations in colorectal cancer (CRC, n = 323) versus all other cancers (Pan-cancer, n = 3396) in TCGA. Shown is the % of cases with each hotspot mutation out of all TP53-mutated cases. Two sided Fisher’s exact test. b Ratio between the numbers of CRC cases with R175 mutations (N = 132) and R273 mutations (N = 121) in stage 1–2 and stage 3–4 disease. Two sided Fisher’s exact test. c Percentage of cases of each mutation type with metastases at uncommon sites (brain, bone, pelvis, peritoneum and omentum) at presentation (N = 66 for R175 tumors and N = 68 for R273 tumors), in the MSKCC cohort. Two sided Fisher’s exact test. d Percentage of cases of each mutation type (N = 66 for R175 tumors and N = 68 for R273 tumors) with multiple metastases (three or more) at presentation, in the MSKCC cohort. Two sided Fisher’s exact test. e Disease specific overall survival of CRC patients with either R175 or R273 mutations. Compiled from TCGA COAD-READ and published data. Log-rank test. f Multivariate Cox regression analysis for the impact of multiple variables on overall survival in the patient collection described in (e). Ovals represent hazard ratios, and error bars (horizontal lines) denote confidence intervals. Source data is provided as a Source Data file.

Fig. 2. R273 mutants orchestrate a distinct transcriptional signature.

a SW480 cells in which the endogenous TP53 genes (harboring R273H and P309S mutations) had been knocked out, were stably transduced with p53^R175H or p53^R273H. b Western blot analysis of p53 in SW480 knockout (KO) cells before and after transduction of p53^R175H or p53^R273H. n = 3. c SW480 TP53 KO cells and their derivatives expressing p53^R175H or p53^R273H were subjected to RNA-seq analysis. Shown is a heatmap of genes differentially expressed (fold change > 1.5, pAdj < 0.05) in p53^R273H overexpressing cells relative to p53 KO and p53^R175H overexpressing cells, n = 3. d Venn diagram of upregulated genes (fold change > 1.5, pAdj < 0.1) in p53^R273H overexpressors relative to p53 KO cells (blue circle) or p53^R175H overexpressors (green circle). The 140 overlapping genes were defined as the ‘R273 signature’. e Western blot analysis of p53 in SW480 cells stably transduced with shRNA directed against the 3’ UTR of the TP53 gene (shp53), followed by stable overexpression of shRNA-resistant p53^R175H or p53^R273H. shc = SW480 cells transduced with control shRNA, to visualize the endogenous p53. n = 3. f, g Gene Set Enrichment Analysis (GSEA) of differentially expressed genes in shp53 cells reconstituted with p53^R273H vs control shp53 cells or shp53 cells reconstituted with p53^R175H (ranked by fold change), using the R273 signature as the tested gene set. ES = Enrichment score. Source data is provided as a Source Data file.

Fig. 3. R273 signature genes are selectively upregulated by p53^R273H in CRC cells.

a HCT116 cells were subjected to CRISPR/Cas 9 gene editing using RNP and ssODN to knock-in either the p53^R175H or the p53^R273H mutation. Cells which underwent the same process but did not end up with an edited genome, and thus retained wtp53 expression, served as CRISPR control. b RT-qPCR analysis of p21 mRNA in the cells in a. For the CRISPR/Cas9 knock-in clones, values in each experiment were determined separately for each individual clone, normalized to GAPDH mRNA, and then averaged. Values in the figure are displayed relative to the control parental cells, defined as 1.0. Mean ± SEM from four independent experiments. one-way ANOVA and Tukey’s post hoc test. c RT-qPCR analysis of representative R273 signature genes in the cells in a. Values were calculated as in b. Three biological repeats (ITGA7,CDC42EP5), four biological repeats (APOE) or five biological repeats (MRC2, ECM1). d Western blot analysis of HT-29 cells transduced with p53-specific shRNA (Shp53) or control shRNA (Shc). n = 2. e Western blot analysis of COGA-5 cells transduced with p53-specific shRNA (Shp53) or control shRNA (Shc). n = 2. f RT-qPCR analysis of representative R273 signature genes in the cells in d. Values were normalized to GAPDH mRNA and are shown relative to the Shc cells. Mean ± SEM from three independent experiments. Unpaired two-tailed t test. g RT-qPCR analysis of representative R273 signature genes in the cells in e. Values were normalized to GAPDH mRNA and are shown relative to the Shc cells. Mean ± SEM from four independent experiments. Unpaired two-tailed t test. Source data is provided as a Source Data file.

Fig. 4. The R273 signature is upregulated in CRC cell lines and tumors and is associated with poor survival.

a Relative expression of the R273 signature in seven CRC cell lines harboring R273 mutations (SW480, SW620, CL14, HT-29, NCIH508, SNU503, SNUC2A) or truncating TP53 mutations (Tr; n = 11). The boxplot displays data quartiles, horizontal lines mark the medians and upper and lower whiskers indicate maximum and minimum values for each distribution. Data accrued from Xena browser Cancer Cell Line Encyclopedia (CCLE) RNA-seq gene expression data (RPKM). Before mean expression calculation, all genes in the R273 signature were normalized to contribute equally to the signature. Unpaired two-tailed t test. b, c GSEA of CRC tumors harboring R273 mutations (n = 28) compared to tumors harboring R175 (n = 36) or truncating (Tr; n = 28) mutations; for truncating mutations, we selected the 28 samples with the lowest p53 mRNA levels, to better approximate null mutations. Genes were ranked by fold change, and the R273 signature was used as the tested gene set. d Pearson R correlation between the R273 signature and the cell-intrinsic gene signatures of the CMS4 subtype (Sveen et al., 2018). e, f GSEA of the same CRC tumors as in b–c, except that the CMS4 gene signature was used as the tested gene set. g Percentage of late-stage (stage 3–4) tumors among CRC tumors in the lowest quartile (N = 173) or highest quartile (N = 174) of R273 signature expression. h Overall survival of patients within the highest or lowest quartile of R273 signature expression in the TCGA colorectal cancer cohort. Log-rank test. Source data is provided as a Source Data file.

Fig. 5. p53^R273H promotes cell spreading, migration and invasion.

a Gene Ontology analysis of the R273 signature (Metascape). b Kinetics of spreading of SW480 p53 KO cells (KO) and their derivatives stably overexpressing p53^R175H or p53^R273H. Percentages of spread cells in the course of 24 h were determined by time-lapse microscopy. Images were taken at 1 h intervals, and were subjected to cell segmentation and aspect ratio calculation. Statistical analysis at t = 24 was done using one-way ANOVA and Tukey’s post hoc test. Two biological repeats. c Representative images of transwell migration assays performed with SW480 p53 KO cells and their derivatives stably overexpressing p53^R175H or p53^R273H, taken 24 h post-seeding. d Average percentage of coverage (ImageJ) by migrating cells in transwell migration assays as described in c. Mean ± SEM from Three biological repeats. Nested one way ANOVA and Tukey’s post hoc test. e Representative images of transwell migration assays performed with HCT116 CRISPR/Cas9 control cells (WT) or CRISPR/Cas9 knock-in of either p53^R175H or p53^R273H. An equal number of cells from each of the 5 clones harboring the same mutation were pooled together and grown for one week prior to the migration assay. f Average percentage of coverage (ImageJ) by migrating cells in transwell migration assays as described in e. Mean ± SEM from four biological repeats. Nested one way ANOVA and Tukey’s post hoc test. g Average percentage of coverage (ImageJ) by invading cells in transwell Matrigel invasion assays performed with the same cells as in c and e. Mean ± SEM from three biological repeats (SW480) or two biological repeats (HCT116). Nested one way ANOVA and adjustment for multiple comparison. h SW480 cells stably overexpressing p53^R175H or p53^R273H were subjected to Rho signaling activation analysis using a G-LISA assay kit. Mean ± SEM from Three technical repeats. i SW480 p53 KO cells stably overexpressing p53^R273H were treated for 4 h with either DMSO or MBQ-167 (750 nM), and then subjected to a transwell migration assay as in c. Average percentage of coverage by migrating cells (ImageJ) is shown. n = 4. Nested one way ANOVA and Tukey’s post hoc test. Source data is provided as a Source Data file.

Fig. 6. p53^R273H preferentially promotes metastasis.

a SW480 p53 KO cells stably overexpressing p53^R175H or p53^R273H were GFP labeled and injected into the tail vein of NSG mice. Lung metastases were visualized nine weeks post-injection. b Total area of metastases at the lung surface (calibrated units), as quantified with ImageJ (Mean ± SEM, 5 mice per group). Two-tailed Mann–Whitney U-test. c Representative images of lung metastases in mice analyzed as in a. d SW480 p53 KO cells stably overexpressing p53^R175H or p53^R273H were injected into the cecal wall of NSG mice. 7 weeks post-injection, Metastases were evaluated by a pathologist, using H&E-stained histology slides. e Numbers of mice with liver, lung, and peritoneal metastases in the groups described in d. f Representative H&E staining images of lung and liver tissue of mice analyzed as in (d). The bottom row shows a 20X magnification of the areas marked by squares in the 5X magnification images in the upper row. Arrows indicate metastatic foci. Source data is provided as a Source Data file.

Fig. 7. p53^R273H binds gene regulatory elements and augments transcription.

a Top five enriched GO cellular components associated with endogenous mutp53 ChIP-seq peaks in SW480 cells. Data from Rahnamoun et al., was subjected to analysis by GREAT as described in Methods. b Mutp53 chromatin binding peaks in SW480 cells are significantly associated with genes upregulated by p53^R273H. All individual genes were ranked by their distance to the nearest p53 ChIP-seq peak in Rahnamoun et al.; the X-axis represents log 10 of the rank. Red line represents the genes upregulated in SW480 TP53 KO cells stably transduced with p53^R273H, relative to control KO cells and cells transduced with p53^R175H (see Fig. 2d). Dashed line indicates all the other, non-differentially expressed genes as background. One tailed Kolmogorov-Smirnov test. c ChIP-qPCR analysis of mutp53 binding to regulatory regions of representative R273 signature genes in SW480 cells stably overexpressing either p53^R175H or p53^R273H. Binding of mutp53 to regulatory elements of ITGA7 and APOE is compared to binding to intronic regions of the same genes. Nested one way ANOVA and Tukey’s post hoc test. Three biological repeats. Total 6 repeats. d RT-qPCR analysis of APOE mRNA in SW480 TP53 KO cells transiently transfected with empty vector control (EV), intact p53^R273H, or p53^R273H harboring two mutations (L22Q and W23S) within the p53 transactivation domain (R273H TAD mutant). Values were normalized to GAPDH mRNA and are shown relative to the empty vector control cells. Mean ± SEM from five independent biological repeats (one-way ANOVA and Tukey’s post hoc test). e Transcription factors (TF) binding sites overrepresented in canonical promotors of the R273 signature genes. Upper panel shows the top 20 TFs enriched in the R273 signature gene promoters relative to all canonical gene promotors. Lower panel shows the extent of overrepresentation of the same 20 TFs, at mutp53 binding sites in SW480 cells, determined experimentally by Rahnamoun et al., relative to the entire human genome sequence. wtp53 is included in both panels as an example of a non-enriched TF. “F” in EGRF, SP1F etc. relates to “family”. Red bars indicate zinc finger transcription factors. Source data is provided as a Source Data file.