R270H And Null Mutant Trp53 Alleles Have Comparable Effects In Regulating Invasion,metastasis And Gene Expression In Mouse Colon Tumorigenesis

Background&Aims:Colorectal cancer is the 2^ndcause of cancer death in global.The incidence and mortality of CRC in China increase every year.The causes of CRCs include environmental and hereditary factors,and great progression have been made in the study of colorectal tumorigenesis.Somatic APC（adenomatous polyposis coli）,TP53,KRAS mutations are the most common mutations in human colorectal cancers and play great roles in the colon adenoma-carcinoma sequence.R273H missense mutation of TP53 is frequently found in human colorectal cancers.In recent years,missense mutant p53 proteins have been reported to exert gain-of-function（GOF）effects in cancer.Here we established genetically engineered mouse CRC models with the mutations in Apc,Kras and Trp53 genes to investigate the potential gain-of-function role of mutant R270H p53 protein plays in CRCs.Methods:Using the CDX2P-Cre ER^T2transgene and Tamoxifen treatment to activate Cre function in colon epithelial cells,we targeted the Apc,Kras and Trp53 alleles in epithelium of the distal ileum,cecum,and colon.In the case,Kras ^LSL-G120Dcould be activated by Cre recombination,while Apc^floxand Trp53^floxcould be inactivated by Cre recombinase function.We generated compound mutant mice with the same Apc^flox/+and Kras ^LSL-G120D/+mutation background,but different Trp53 mutant alleles combinations.We compared the difference in life span,tumor burden,tumor histology,the depth of tumor invasion,the frequencies of spontaneous metastasis to lymph nodes,lung and liver,the expression of epithelial-mesenchymal transition（EMT）associated molecule makers,E-cadherin and vimentin in different mice groups,to investigate the role of mutant p53 in the invasion and metastasis of CRCs.Based on the genetically engineered mouse CRC models,we generated the corresponding colon carcinomas derived organoids.Using DNA chips,we compared the global gene expression difference between carcinomas tissues（or tumor-derived organoids）with Trp53 R270H or null mutation.Along with the analysis of RNA-seq data for CRCs in The Cancer Genome Atlas（TCGA）,we aimed to find the potential effect of R270H/R273 mutation on the global gene expression profiles of CRCs.Results:We successfully introduced mutant Trp53 alleles into CDX2P-Cre ER^T2Apc^flox/+Kras ^LSL-G120D/+（AK）mice,so established 3 types ofgeneticallyengineeredmouseCRCmodels:CDX2P-Cre ER^T2Apc^flox/+Kras ^LSL-G120D/+Trp53^270/+(AKP^270/+),CDX2P-Cre ER^T2Apc^flox/+Kras ^LSL-G120D/+Trp53^R270H/flox(AKP^270/fl),and CDX2P-Cre ER^T2Apc^flox/+Kras ^LSL-G120D/+Trp53^flox/flox(AKP^fl/fl)mice.We found that addition of a Trp53^R270Hor Trp53^nullmutant allele to the model（AKP mice）led to markedly shortened survival and increased tumor burden relative to that of AK mice,including adenocarcinomas in AKP mice.But AKP^270/fland AKP^fl/flmice have comparable life span and tumor burden,similar tumor histology and EMT features,along with similar frequencies of spontaneous metastasis to lymph nodes,lung,and liver.The fraction of adenocarcinomas with submucosa or deeper invasion was higher in AKP^270/flmice than in AKP^fl/flmice（36.9%VS26.1%;P=0.0026）,but the incidence of adenocarcinomas per mouse did not differ significantly between AKP^fl/fland AKP^270/flmice（8.9 VS 7.3 per mouse,P=0.16）.In line with their comparable biological behaviors,mouse primary tumors and tumor-derived organoids with the Trp53^R270Hor Trp53^nullalleles had highly similar gene expression profiles.Human CRCs with TP53 R273 missense mutant or null alleles also had essentially homogeneous gene expression patterns.Conclusions:Trp53 null and R270H mutant alleles have comparable effects in promoting invasion,metastasis in mouse colon tumorigenesis,but the introduction of the 2 mutant alleles doesn’t make significant difference in the global gene expression profiles of CRCs.Our findings indicate the R270H/R273 p53 mutant protein does not manifest definite GOF biological effects in mouse and human CRCs,suggesting possible GOF effects of mutant p53 in cancer phenotypes are likely allele-specific and/or context-dependent.